Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.     

<Emphasis Type="Italic">Thermobaculum terrenum</Emphasis> gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65–92 °C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 °C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1^T (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).     

Update on the genetics of migraine

The field of migraine genetics has seen an explosion of information over the last year. In a recent breakthrough, missense mutations in a chromosome 1q23 gene, ATP1A2, encoding a Na⁺, K⁺-ATPase, have been identified in four distinct pedigrees with a rare form of familial hemiplegic migraine (FHM). ATP1A2 is expressed in the brain, like the voltage gated calcium channel gene, CACNA1A, previously identified as the first hemiplegic migraine gene (FHM1). The shared hemiplegic migraine phenotype of mutations in ATP1A2 and CACNA1A raises the possibility that they coordinately regulate ion homeostasis that determines susceptibility to the initiation of both migraine aura and the pain phase of migraine. For the more common and genetically complex forms of migraine, genome-wide screens have identified several new loci on 4q24, 6p12.2–21.1, 11q24, and 14q21.2-q22.3, suggesting additional migraine genes in these regions. In addition, a recent large case-control association study has linked single nucleotide polymorphisms in the insulin receptor/INSR gene with migraine. However, these polymorphisms do not result in detectable changes in receptor function. The continuing genetic identification of key proteins involved in migraine will refine our understanding of this common and sometimes debilitating disorder, which can strike during the most productive years of a persons life. Given the co-morbidity of migraine with depression and bipolar disorder, our knowledge of the causes of migraine may also contribute to our understanding of these disorders.     

Identification of stathmin as a novel marker of cell proliferation in the recovery phase of acute ischemic renal failure

Ischemic renal injury can be classified into the initiation and extension phase followed by the recovery phase. The recovery phase is characterized by increased dedifferentiated and mitotic cells in the damaged tubules. Suppression subtractive hybridization was performed by using RNA from normal and ischemic kidneys to identify the genes involved in the physiological response to ischemia-reperfusion injury (IRI). The expression of stathmin mRNA increased by fourfold at 24 h of reperfusion. The stathmin mRNA did not increase in sodium-depleted animals or in animals with active, persistent injury secondary to cis-platinum. Immunofluorescent labeling demonstrated that the expression of stathmin increased dramatically at 48 h of reperfusion. Labeling with antibodies to stathmin and proliferating cell nuclear antigen (PCNA) indicates that the expression of stathmin was induced before the upregulation of PCNA and that all PCNA-positive cells expressed stathmin. Double immunofluorescent labeling demonstrated the colocalization of stathmin with vimentin, a marker of dedifferentiated cells. Stathmin expression was also significantly enhanced in acute tubular necrosis in humans. On the basis of its induction profile in IRI, the data indicating its enhanced expression in proliferating cells and regenerating organs, we propose that stathmin is a marker of dedifferentiated, mitotically active epithelial cells that may contribute to tubular regeneration and could prove useful in distinguishing the injury phase from recovery phase in IRI.     

Lipid rafts in cytokine signaling

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.     

Coping mechanisms of alpine and arctic breeding birds: extreme weather and limitations to reproductive resilience

As ground nesting homeotherms, alpine and arctic birds mustmeet similar physiological requirements for breeding as otherbirds, but must do so in more extreme conditions. Annual springsnowfall and timing of snow melt can vary by up to 1 month anddaily temperatures near the ground surface vary from below freezingto over 45°C in alpine and arctic habitats. Species breedingin these environments have various behavioral, physiological,and morphological adaptations to cope with energetically demandingconditions. We review the ways birds cope with harsh and variableweather, and present data from long term field studies of ptarmiganto examine effects of spring weather on reproduction. In variablebut normal spring conditions, timing of breeding was not influencedby snow melt, snow depth or daily temperatures in the alpine,as breeding did not commence until conditions were generallyfavorable. Arctic ptarmigan tended to vary breeding onset inresponse to spring conditions. Generally, birds breeding inalpine and arctic habitats suffer a seasonal reproductive disadvantagecompared to birds at lower latitudes or elevations because thebreeding window is short and in late years, nest failure maybe high with little opportunity for renesting. Coping mechanismsmay only be effective below a threshold of climactic extremes.Despite strong resilience in fecundity parameters, when snowmeltis extremely delayed breeding success is greatly reduced. Alpineand arctic birds will be further challenged as they attemptto cope with anticipated increases in the frequency and severityof weather events (climate variability), as well as generalclimate warming.     

Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr

The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.     

A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

Background

The erythrocyte binding antigen-175 (EBA-175) on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. Dimorphic allele segments have been found in its encoding gene with a 342 bp segment present in FCR-3 strains (F-segment) and a 423 bp segment in CAMP strains (C-segment). Possible associations of the dimorphism with severe malaria have been analysed in a case-control study in northern Ghana.

Methods

Blood samples of 289 children with severe malaria and 289 matched parasitaemic but asymptomatic controls were screened for eba- 175 F- and C-segments by nested polymerase chain reaction.

Results

In children with severe malaria, prevalences of F-, C- and mixed F-/C-segments were 70%, 19%, and 11%, respectively. The C-segment was found more frequently in severe malaria cases whereas mixed infections were more common in controls. Infection with strains harbouring the C-segment significantly increased the risk of fatal outcome.

Conclusion

The results show that the C-segment is associated with fatal outcome in children with severe malaria in northern Ghana, suggesting that it may contribute to the virulence of the parasite.     

Model of angiogenesis in mice with severe combined immunodeficiency (SCID) and xenoengrafted with Epstein-Barr virus-transformed B cells

Xenoengraftment of human cells in mice with severe combined immunodeficiency (SCID) has been used as a model system to study the mechanisms of B-cell lymphomagenesis. In the study reported here, we determined that SCID mice can also be used as a model to study angiogenesis in B-cell lymphomas. The C.B-17 scid/scid mice were xenotransplanted with Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), and we determined whether CD31, a marker found on endothelial cells, was detected in the human B-cell lymphomas that developed in these mice. Microvessel formation was identified by use of immunohistochemical staining for CD31. To assess possible mechanisms of angiogenic stimulus, we analyzed the expression of interleukin 8 (IL-8), a chemokine documented to promote angiogenesis, in non-small-cell lung cancer and bronchogenic carcinomas. We observed that a panel of LCL and LCL-lymphomas expressed IL-8 mRNA and protein. Neutralization of IL-8, however, did not inhibit lymphomagenesis, suggesting that IL-8 is not essential for angiogenesis in this model. To examine other parameters of angiogenesis, we identified expression of vascular endothelial growth factor in the lymphomas. These data suggest that angiogenesis accompanies EBV-associated B-cell lymphoma development, but IL-8 is not essential for this process. Thus, the SCID mouse model is amenable to testing of anti-angiogenic factors.