Phytoplasmas ('Candidatus Phytoplasma' spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of 'Ca.Phytoplasma' spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from 'Ca.Phytoplasma' spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.
Studies have suggested that apoptosis may contribute to the neuronal cell loss observed in Alzheimer's disease (AD). Aβ 1–42 has been shown to induce apoptosis in neurons and may be an initiating factor in AD. Caspase is an effector in neuronal apoptosis that could also play a role in AD. However, the extent to which apoptosis contributes to cell death in AD has yet to be delineated. In an earlier study, we identified and isolated Chlamydia pneumoniae from brains of patients that had been diagnosed with sporadic AD. These in vitro studies suggested that neurons infected with C. pneumoniae are resistant to apoptosis, and that the processing or production of APP into Aβ1–42 was increased by the infection. In addition, we have developed a novel murine model in which non–transgenic mice infected with C. pneumoniae formed deposits of amyloid in areas of the brain most affected in Alzheimer's disease. Interestingly, some of the neurons in these areas showed a high level of Aβ 1–42 immunoreactivity, and these neurons did not appear to be undergoing apoptosis. The focus of the current studies was to delineate whether caspase is activated following a C. pneumoniae infection in neuronal cells. Apoptosis was experimentally induced by staurosporine in uninfected SK–N–MC cells and cells infected with C. pneumoniae. Caspase activity was analyzed using the Apo–ONE caspase 3/7 assay (Promega). We found that staurosporine induced an increase in caspase 3/7 activity in both infected and uninfected cells, however the staurosporine–treated infected cells had lower activity than even the basal activity of uninfected cells. These data were consistent with immunocytochemistry, which showed decreased labeling by antibodies that recognize cleaved (active) caspase 3 in the infected cells. These results suggest that inhibition of apoptosis by suppression of caspase 3/7 activity, and/or by decreasing levels of active caspase 3, may be mechanisms by which C. pneumoniae can sustain a persistent infection in the host and optimize its intracellular environment. In this way, C. pneumoniae may participate in the pathogenesis characteristic of Alzheimer's disease.
Sporadic late–onset Alzheimer's disease is a non–familial, progressive neurodegenerative disease associated with atrophy and death of nerve cells in affected brain regions, yet very little is known concerning the cause(s) of sporadic AD. In this regard, our studies have focused on pathogen involvement as a risk or causative factor in sporadic AD. Since we first identified Chlamydophila (Chlamydia) pneumoniae in the AD brain (Balin et al., 1998), there has been controversy surrounding whether this organism is involved in AD pathogenesis. This controversy stems, in part, from the absence of standardized molecular and immunolabeling approaches consistent between laboratories. In this regard, a recent study using molecular approaches for the detection of C. pneumoniae in AD confirms portions of our earlier work (Gerard et al., 2006). The focus of the current investigation is to immunolabel AD brain tissues for C. pneumoniae using commercially available antibody probes to determine the extent of this infection in AD. On sections from AD brains, we used a battery of anti–chlamydial antibodies to determine the relationship of C. pneumoniae to AD neuropathology. Monoclonal and polyclonal antibodies used in this study included: those generated against organisms in the acute vs chronic/persistent stage of infection, those specific to the organism in the elementary or reticulate body stage of development, and those generated against a specific antigen (eg, outer membrane protein vs lipopolysaccharide). We observed differential immunolabeling patterns for the detection of C. pneumoniae. The patterns of labeling consisted of intracellular labeling of neuronal, glial, and endothelial cells, as well as diffuse extracellular labeling in regions of neuropathology. Intriguingly, antibodies that labeled in a diffuse extracellular manner, at times, immunolabeled within amyloid beta 1–42 immunoreactive plaques. Our data suggest that evidence for C. pneumoniae infection is present in areas of neuropathology in sporadic AD brain tissues, that some antigens from C. pneumoniae may colocalize or interact with beta amyloid, and that a battery of antibodies must be used to detect both the intracellular and extracellular presence of C. pneumoniae in the AD brain.
Sporadic, late-onset Alzheimer's disease (LOAD) is a non-familial, progressive neurodegenerative disease that is now the most common and severe form of dementia in the elderly. That dementia is a direct result of neuronal damage and loss associated w
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