Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

Background

Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia.

Methodology/Principal Findings

The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int.

Conclusions/Significance

Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.     

Detection of bradykinin and bradykinin-beta(2) receptors in the porcine endometrium during the estrous cycle and early pregnancy

During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin beta(2) receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12-18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12-18 of pregnancy compared with the estrous cycle. Endometrial bradykinin beta(2) receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin beta(2) receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.     

HGF converts ErbB2/Neu epithelial morphogenesis to cell invasion

Activation of the hepatocyte growth factor receptor Met induces a morphogenic response and stimulates the formation of branching tubules by Madin-Darby canine kidney (MDCK) epithelial cells in three-dimensional cultures. A constitutively activated ErbB2/Neu receptor, NeuNT, promotes a similar invasive morphogenic program in MDCK cells. Because both receptors are expressed in breast epithelia, are associated with poor prognosis, and hepatocyte growth factor (HGF) is expressed in stroma, we examined the consequence of cooperation between these signals. We show that HGF disrupts NeuNT-induced epithelial morphogenesis, stimulating the breakdown of cell-cell junctions, dispersal, and invasion of single cells. This correlates with a decrease in junctional proteins claudin-1 and E-cadherin, in addition to the internalization of the tight junction protein ZO-1. HGF-induced invasion of NT-expressing cells is abrogated by pretreatment with a pharmacological inhibitor of the mitogen-activated protein kinase kinase (MEK) pathway, which restores E-cadherin and ZO-1 at cell-cell junctions, establishing the involvement of MEK-dependent pathways in this process. These results demonstrate that physiological signals downstream from the HGF/Met receptor synergize with ErbB2/Neu to enhance the malignant phenotype, promoting the breakdown of cell-cell junctions and enhanced cell invasion. This is particularly important for cancers where ErbB2/Neu is overexpressed and HGF is a physiological growth factor found in the stroma.     

Murine mCLCA6 is an integral apical membrane protein of non-goblet cell enterocytes and co-localizes with the cystic fibrosis transmembrane conductance regulator.

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine.     

Quorum sensing contributes to natural transformation of Vibrio cholerae in a species-specific manner

Although it is a human pathogen, Vibrio cholerae is a regular member of aquatic habitats, such as coastal regions and estuaries. Within these environments, V. cholerae often takes advantage of the abundance of zooplankton and their chitinous molts as a nutritious surface on which the bacteria can form biofilms. Chitin also induces the developmental program of natural competence for transformation in several species of the genus Vibrio. In this study, we show that V. cholerae does not distinguish between species-specific and non-species-specific DNA at the level of DNA uptake. This is in contrast to what has been shown for other Gram-negative bacteria, such as Neisseria gonorrhoeae and Haemophilus influenzae. However, species specificity with respect to natural transformation still occurs in V. cholerae. This is based on a positive correlation between quorum sensing and natural transformation. Using mutant-strain analysis, cross-feeding experiments, and synthetic cholera autoinducer-1 (CAI-1), we provide strong evidence that the species-specific signaling molecule CAI-1 plays a major role in natural competence for transformation. We suggest that CAI-1 can be considered a competence pheromone.     

Oleuropein and derivatives from olives as Tau aggregation inhibitors

Tau isoforms constitute a family of microtubule-associated proteins that are mainly expressed in neurons of the central nervous system. They promote the assembly of tubulin monomers into microtubules and modulate their stability, thus playing a key structural role in the distal portion of axons. In Alzheimer's disease and related tauopathies, Tau aggregation into fibrillary tangles contributes to intraneuronal and glial lesions. We report herein the ability of three natural phenolic derivatives obtained from olives and derived food products to prevent such Tau fibrillization in vitro, namely hydroxytyrosol, oleuropein, and oleuropein aglycone. The latter was found to be more active than the reference Tau aggregation inhibitor methylene blue on both wild-type and P301L Tau proteins, inhibiting fibrillization at low micromolar concentrations. These findings might provide further experimental support for the beneficial nutritional properties of olives and olive oil as well as a chemical scaffold for the development of new drugs aiming at neurodegenerative tauopathies.     

IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

Validation of Cell-Cycle Arrest Biomarkers for Acute Kidney Injury after Pediatric Cardiac Surgery

Background

The lack of early biomarkers for acute kidney injury (AKI) seriously inhibits the initiation of preventive and therapeutic measures for this syndrome in a timely manner. We tested the hypothesis that insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, function as early biomarkers for AKI after congenital heart surgery with cardiopulmonary bypass (CPB).

Methods

We prospectively studied 51 children undergoing cardiac surgery with CPB. Serial urine samples were analyzed for [TIMP-2]•[IGFBP7]. The primary outcome measure was AKI defined by the pRIFLE criteria within 72 hours after surgery.

Results

12 children (24%) developed AKI within 1.67 (SE 0.3) days after surgery. Children who developed AKI after cardiac surgery had a significant higher urinary [TIMP-2]•[IGFBP7] as early as 4 h after the procedure, compared to children who did not develop AKI (mean of 1.93 ((ng/ml)²/1000) (SE 0.4) vs 0.47 ((ng/ml)²/1000) (SE 0.1), respectively; p<0.05). Urinary [TIMP-2]•[IGFBP7] 4 hours following surgery demonstrated an area under the receiver-operating characteristic curve of 0.85. Sensitivity was 0.83, and specificity was 0.77 for a cutoff value of 0.70 ((ng/ml)²/1000).

Conclusions

Urinary [TIMP-2]•[IGFBP7] represent sensitive, specific, and highly predictive early biomarkers for AKI after surgery for congenital heart disease.

Trial Registration

www.germanctr.de/, DRKS00005062     

Effects of landscape matrix on population connectivity of an arboreal mammal,Petaurus breviceps

Ongoing habitat loss and fragmentation is considered a threat to biodiversity as it can create small, isolated populations that are at increased risk of extinction. Tree‐dependent species are predicted to be highly sensitive to forest and woodland loss and fragmentation, but few studies have tested the influence of different types of landscape matrix on gene flow and population structure of arboreal species. Here, we examine the effects of landscape matrix on population structure of the sugar glider (Petaurus breviceps) in a fragmented landscape in southeastern South Australia. We collected 250 individuals across 12 native Eucalyptus forest remnants surrounded by cleared agricultural land or exotic Pinus radiata plantations and a large continuous eucalypt forest. Fifteen microsatellite loci were genotyped and analyzed to infer levels of population differentiation and dispersal. Genetic differentiation among most forest patches was evident. We found evidence for female philopatry and restricted dispersal distances for females relative to males, suggesting there is male‐biased dispersal. Among the environmental variables, spatial variables including geographic location, minimum distance to neighboring patch, and degree of isolation were the most important in explaining genetic variation. The permeability of a cleared agricultural matrix to dispersing gliders was significantly higher than that of a pine matrix, with the gliders dispersing shorter distances across the latter. Our results added to previous findings for other species of restricted dispersal and connectivity due to habitat fragmentation in the same region, providing valuable information for the development of strategies to improve the connectivity of populations in the future.