Mechanisms underlying the evolution and maintenance of functionally heterogeneous 18S rRNA genes in Apicomplexans

In many species of the protist phylum Apicomplexa, ribosomal RNA (rRNA) gene copies are structurally and functionally heterogeneous, owing to distinct requirements for rRNA-expression patterns at different developmental stages. The genomic mechanisms underlying the maintenance of this system over long-term evolutionary history are unclear. Therefore, the aim of this study was to investigate what processes underlie the long-term evolution of apicomplexan 18S genes in representative species. The results show that these genes evolve according to a birth-and-death model under strong purifying selection, thereby explaining how divergent 18S genes are generated over time while continuing to maintain their ability to produce fully functional rRNAs. In addition, it was found that Cryptosporidium parvum undergoes a rapid form of birth-and-death evolution that may facilitate host-specific adaptation, including that of type I and II strains found in humans. This represents the first case in which an rRNA gene family has been found to evolve under the birth-and-death model.     

A novel proteomic coculture model of prostate cancer cell growth

Chemotherapy and androgen ablation therapy are only temporarily effective against prostate cancer, and current studies are ongoing to test agents that target proteins responsible for autocrine and paracrine stimulated growth. Given limitations of current laboratory models to test the effect of these agents on cell growth and protein targets, we developed a coculture model that can distinguish paracrine stimulated growth and effects on proteins. We found that LNCaP prostate cancer cells and an immortalized rat prostate cell line transfected to overexpress the antiapoptotic resistance protein Bcl-2 were stimulated to grow (>2-fold increase, p < 0.01) through autocrine effects from additional cells in an upper chamber of our system. Using a proteomic approach with a two-dimensional differential in gel electrophoresis method to increase fidelity, four proteins were found to increase after autocrine induced growth stimulation. These proteins were all identified by mass spectrometry as enzymes in the glycolytic pathway, validating the ability of this system to detect both clonogenic growth and the effect on proteins. These data, therefore, demonstrate a novel coculture model for further study of agents that target proteins in pathways of paracrine or autocrine stimulated cell growth.     

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C10 alkylamines to produce C10-PEAA. Incubation of C10-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C10-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C10-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C10-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C10-PEAA-LUV. It is concluded that C10-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.     

Altered histology of the thymus and spleen in contaminant-exposed juvenile American alligators

Morphological differences in spleen and thymus are closely related to functional immune differences. Hormonal regulation of the immune system has been demonstrated in reptilian splenic and thymic tissue. Spleens and thymus were obtained from juvenile alligators at two reference sites in Florida, USA: Orange Lake and a National Wildlife Refuge, Lake Woodruff, as well as from a contaminated lake, Lake Apopka. Lake Apopka has been extensively polluted with agricultural pesticides. Tissues were prepared for histological analysis to determine if previously detected endocrine abnormalities associated with contaminant exposure might also be reflected in morphological differences in splenic and thymic structures important for immunological response. Similar tissues were taken from captive-raised juvenile female alligators (3 years old) that were hatched from eggs collected on Lake Woodruff and Lake Apopka. Differences in thymic ratios (medulla/cortex) were found among alligators collected from the two lakes (P = 0.0051). Alligators from Lake Apopka had smaller thymic ratios than animals from either reference lake. Males from Lake Woodruff had significantly smaller lymphocyte sheaths in the spleen than females (P = 0.0009), indicative of a normal sexual dimorphism. Lymphocyte sheath width differed among females obtained from the three lakes, with females from Lake Apopka having the smallest sheath width and those from Orange Lake having the largest. Malpighian body area was largest in alligators from Orange Lake, intermediate in Lake Woodruff, and smallest in Lake Apopka. In contrast to that observed for wild-caught animals, no difference was found in the thymic medulla/cortex ratio of captive-raised female alligators (P = 0.378). Captive-raised female alligators from Lake Apopka and Lake Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals; Lake Apopka alligators had smaller lymphocyte sheath width compared to Woodruff alligators (P = 0.0396). In contrast to wild-caught animals, area of the Malpighian bodies did not differ by lake in the captive-raised female alligators (P = 0.066). The enlarged thymic cortex suggests a change in T-lymphocyte maturation within the thymus of alligators from a contaminated environment, Lake Apopka. The results point to alterations in the histology of the thymus and spleen. Further studies are required to examine the functional significance of these observations.     

Genetic consequences of white-tailed deer (Odocoileus virginianus) restoration in Mississippi

White-tailed deer (Odocoileus virginianus) were nearly extirpated from the southeastern USA during the late 19th and early 20th centuries. Recovery programmes, including protection of remnant native stocks and transplants from other parts of the species' range, were initiated in the early 1900's. The recovery programmes were highly successful and deer are presently numerous and continuously distributed throughout the southeastern USA. However, the impact of the recovery programmes on the present genetic structure of white-tailed deer remains to be thoroughly investigated. We used 17 microsatellite DNA loci to assess genetic differentiation and diversity for 543 white-tailed deer representing 16 populations in Mississippi and three extra-state reference populations. There was significant genetic differentiation among all populations and the majority of genetic variation (> or = 93%) was contained within populations. Patterns of genetic structure, genetic similarity and isolation by distance within Mississippi were not concordant with geographical proximity of populations or subspecies delineations. We detected evidence of past genetic bottlenecks in nine of the 19 populations examined. However, despite experiencing genetic bottlenecks or founder events, allelic diversity and heterozygosity were uniformly high in all populations. These exceeded reported values for other cervid species that experienced similar population declines within the past century. The recovery programme was successful in that deer were restored to their former range while maintaining high and uniform genetic variability. Our results seem to confirm the importance of rapid population expansion and habitat continuity in retaining genetic variation in restored populations. However, the use of diverse transplant stocks and the varied demographic histories of populations resulted in fine-scale genetic structuring.     

Fitness reduction and potential extinction of wild populations of Atlantic salmon, Salmo salar, as a result of interactions with escaped farm salmon

The high level of escapes from Atlantic salmon farms, up to two million fishes per year in the North Atlantic, has raised concern about the potential impact on wild populations. We report on a two-generation experiment examining the estimated lifetime successes, relative to wild natives, of farm, F(1) and F(2) hybrids and BC(1) backcrosses to wild and farm salmon. Offspring of farm and "hybrids" (i.e. all F(1), F(2) and BC(1) groups) showed reduced survival compared with wild salmon but grew faster as juveniles and displaced wild parr, which as a group were significantly smaller. Where suitable habitat for these emigrant parr is absent, this competition would result in reduced wild smolt production. In the experimental conditions, where emigrants survived downstream, the relative estimated lifetime success ranged from 2% (farm) to 89% (BC(1) wild) of that of wild salmon, indicating additive genetic variation for survival. Wild salmon primarily returned to fresh water after one sea winter (1SW) but farm and 'hybrids' produced proportionately more 2SW salmon. However, lower overall survival means that this would result in reduced recruitment despite increased 2SW fecundity. We thus demonstrate that interaction of farm with wild salmon results in lowered fitness, with repeated escapes causing cumulative fitness depression and potentially an extinction vortex in vulnerable populations.     

GABA-induced neurite outgrowth of cerebellar granule cells is mediated by GABA(A) receptor activation,calcium influx and CaMKII and erk1/2 pathways

During neuronal development, GABAA-mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1-1000 microm) induces an increase in the intracellular calcium concentration through the activation of GABA(A) receptors and voltage-gated calcium channels of the L-subtype. Perforated patch-clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to - 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro. On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium-dependent mechanism triggered by voltage-gated calcium channel activation. Specific blockers of calcium-calmodulin kinase II and mitogen-activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium-dependent kinases.     

Induction of antigen-specific regulatory T cells following overexpression of a Notch ligand by human B lymphocytes

In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4(+)CD25(+) and CD8(+)CD25(-) T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 (CD39L2) nucleotidase: implications for structure and function

The ectonucleoside triphosphate diphosphohydrolases (NTPDases) control extracellular nucleotide concentrations, thereby modulating many important biological responses, including blood clotting and pain perception. NTPDases1-4 are oligomeric integral membrane proteins, whereas NTPDase5 (CD39L4) and NTPDase6 (CD39L2) are soluble monomeric enzymes, making them more amenable to thorough structural and functional analyses than the membrane-bound forms. Therefore, we report here the bacterial expression, refolding, purification, and biochemical characterization of the soluble portion of human NTPDase6. Consistent with the enzyme expressed in mammalian cells, this recombinant NTPDase6 efficiently hydrolyzes GDP, IDP, and UDP (specific activity of approximately 50000 micromol mg(-1) h(-1)), with slower hydrolysis of CDP, ITP, GTP, CTP, ADP, and UTP and virtually no hydrolysis of ATP. The K(m) for GDP (130 +/- 30 microM) is similar to that determined for the soluble rat NTPDase6 expressed in mammalian cells. The secondary structure of the refolded enzyme was determined by circular dichroism to be 33% alpha-helix, 18% beta-sheet, and 49% random coil, consistent with the secondary structure predicted from the amino acid sequence of soluble NTPDase6. Four of the five cysteine residues in the soluble NTPDase6 are highly conserved among all the NTPDases, while the fifth residue is not. Mutation of this nonconserved cysteine resulted in an enzyme very similar to wild type in its enzymology and secondary structure, indicating that this cysteine exists as a free sulfhydryl and is not essential for structure or function. The disulfide pairing of the other four cysteine residues was determined as Cys(249)-Cys(280) and Cys(340)-Cys(354) by HPLC and mass spectral analysis of tryptic peptides. Due to conservation of these cysteine residues, these two disulfide bonds are likely to exist in all NTPDases. A structural model for NTPDase6, incorporating these and other findings obtained with other NTPDases, is proposed.