Life history focus on a malaria parasite: linked traits and variation among genetic clones

Life history theory has long been a major campaign in evolutionary ecology, but has typically focused only on animals and plants. Life history research on single-celled eukaryotic protists such as malaria parasites (Plasmodium) will offer new insights into the theory’s general utility as well as the parasite’s basic biology. For example, parasitologists have described the Plasmodium life cycle and cell types in exquisite detail, with little discussion of evolutionary issues such as developmental links between traits. We measured 10 life history traits of replicate single-genotype experimental Plasmodium mexicanum infections in its natural lizard host to identify groups of linked traits. These 10 traits formed 4 trait groups: “Rate/Peak” merges measures of growth rate and maximum parasitemia of infections; “Timing” combines time to patency and maximum parasitemia; “Growth Shape” describes the fit to an exponential growth curve; and “Sex Ratio” includes only the gametocyte sex ratio. Parasite genotype (clone) showed no effect on the life history trait groups, with the exception of gametocyte sex ratio. Therefore, variation in most life history traits among infections appears to be driven by environmental (individual host) effects. The findings support the model that life history traits are often linked by developmental constraints. Understanding why life history traits of Plasmodium are linked in this way would offer a new window into the evolution of the parasites, and also should inform public health efforts to control infection prevalence.     

Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA

DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA. The temperature dependence of the binding affinity exhibits distinct curvature, with tightest binding at 25-30 degrees C. Nonlinear temperature dependence indicates Klenow binds different primed-template constructs with large heat capacity (DeltaCp) values (-870 to -1220 cal/mole K) and thus exhibits large temperature dependent changes in enthalpy and entropy. Binding is entropy driven at lower temperatures and enthalpy driven at physiological temperatures. Large negative DeltaCp values have been proposed to be a 'signature' of site-specific DNA binding, but type I DNA polymerases do not exhibit significant DNA sequence specificity. We suggest that the binding of Klenow to a specific DNA structure, the primed-template junction, results in a correlated thermodynamic profile that mirrors what is commonly seen for DNA sequence-specific binding proteins. Klenow joins a small number of other DNA-sequence independent DNA binding proteins which exhibit unexpectedly large negative DeltaCp values. Spectroscopic measurements show small conformational rearrangements of both the DNA and Klenow upon binding, and small angle x-ray scattering shows a global induced fit conformational compaction of the protein upon binding. Calculations from both crystal structure and solution structural data indicate that Klenow DNA binding is an exception to the often observed correlation between DeltaCp and changes in accessible surface area. In the case of Klenow, surface area burial can account for only about half of the DeltaCp of binding.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

Biosynthesis of α-Ketoglutarate by the Reductive Carboxylation of Succinate in Bacteroides ruminicola

Experiments with growing cells and with cell-free extracts of Bacteroides ruminicola indicate that this anaerobic bacterium can synthesize alpha-ketoglutarate by a reductive carboxylation of succinate. When the organism was grown in medium containing succinate-1,4-(14)C, most of the radioactivity in cells was in the protein fraction and most of the (14)C in protein was in the glutamic acid family of amino acids (glutamate, proline, and arginine). When unlabeled succinate was added to culture medium containing glucose-U-(14)C, incorporation of radioactivity into the glutamic acid family of amino acids was greatly reduced. This supports the concept that succinate is an intermediate in synthesis of alpha-ketoglutarate. Cell-free extracts of the organism incubated with succinate-1,4-(14)C incorporated (14)C into amino acids and most of this was found in glutamate. The cofactors which stimulate glutamate synthesis from succinate by extracts from these cells appear to be similar to the factors that have been demonstrated with extracts from photosynthetic bacteria. The position of label in glutamate synthesized from succinate-1,4-(14)C, the probable absence of isocitric dehydrogenase, and studies with labeled citrate and with inhibitors of citric acid cycle enzymes support the concept of a reductive carboxylation of succinate as the only, or at least a major, mechanism for synthesis of alpha-ketoglutarate in this organism. This appears to be the first evidence for a net synthesis of alpha-ketoglutarate by this reaction in a nonphotosynthetic heterotrophic organism.     

Expression of <Emphasis Type="Italic">PEG11</Emphasis> and <Emphasis Type="Italic">PEG11AS</Emphasis> transcripts in normal and callipyge sheep

Background  

The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression.     

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression

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Regulation of the type III InsP(3) receptor by InsP(3) and calcium

It has been proposed that the inositol 1,4,5-trisphosphate receptor (InsP(3)R) type III acts as a trigger for InsP(3)-mediated calcium (Ca(2+)) signaling, because this InsP(3) isoform lacks feedback inhibition by cytosolic Ca(2+). We tested this hypothesis in RIN-m5F cells, which express predominantly the type III receptor. Extracellular ATP increases Ca(2+) in these cells, and we found that this effect is independent of extracellular Ca(2+) but is blocked by the InsP(3)R antagonist heparin. There was a dose-dependent increase in the number of cells responding to ATP and two-photon flash photolysis of caged-Ca(2+) heightened the sensitivity of RIN-m5F cells to this increase. These findings provide evidence that Ca(2+) increases the sensitivity of the InsP(3)R type III in intact cells and supports the idea that this isoform can act as a trigger for hormone-induced Ca(2+) signaling.     

Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome

It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-alpha, IFN-gamma, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.     

Geographic patterns of genetic variation in native pecans

A structured collection of 80 seedling pecan trees [Carya illinoinensis (Wangenh.) K. Koch], representing 19 putatively native pecan populations across the species range, was evaluated at three plastid and 14 nuclear microsatellite (simple sequence repeat, SSR) loci. Data were analyzed using a priori population designations and also within a Bayesian framework, in which individuals were assigned to clusters regardless of population of origin. Population genetic analyses using a priori populations, clusters based on chloroplast microsatellite data (cpSSR), and clusters based on nuclear microsatellite data (nSSR) yielded consistent results. For all groupings, cpSSR variation exhibited more geographic structure than the nSSR data. Furthermore, cpSSR microsatellite diversity decreased with increasing latitude, but this pattern was not observed with the nuclear data. Contrasting patterns in plastid and nuclear genetic diversity demonstrate unique aspects of postglacial recolonization reflected in the movement of seeds versus pollen. These data suggest that plastid SSRs are useful tools for identifying population structure in pecan and hold promise for ongoing efforts to identify and conserve representative germplasm in ex situ collections.