Peptidyl-prolyl cis-trans isomerases (immunophilins) and their roles in parasite biochemistry, host-parasite interaction and antiparasitic drug action.

Immunophilin is the collective name given to the cyclophilin and FK506-binding protein (FKBP) families. As the name suggests, these include the major binding proteins of certain immunosuppressive drugs: cyclophilins for the cyclic peptide cyclosporin A and FKBPs for the macrolactones FK506 and rapamycin. Both families, although dissimilar in sequence, possess peptidyl-prolyl <i>cis-trans</i> isomerase activity in vitro and can play roles in protein folding and transport, RNA splicing and the regulation of multiprotein complexes in cells. In addition to enzymic activity, many immunophilins act as molecular chaperones. This property may be conferred by the isomerase domain and/or by additional domains. Recent years have seen a great increase in the number of known immunophilin genes in parasitic protozoa and helminths and in many cases their products have been characterized biochemically and their temporal and spatial expression patterns have been examined. Some of these genes represent novel types: one example is a <i>Toxoplasma gondii</i> gene encoding a protein with both cyclophilin and FKBP domains. Likely roles in protein folding and oligomerisation, RNA splicing and sexual differentiation have been suggested for parasite immunophilins. In addition, unexpected roles in parasite virulence (Mip FKBP of <i>Trypanosoma cruzi</i>) and host immuno-modulation (e.g. 18-kDa cyclophilin of <i>Toxoplasma gondii</i>) have been established. Furthermore, in view of the potent antiparasitic activities of cyclosporins, macrolactones and nonimmunosuppressive derivatives of these compounds, immunophilins may mediate drug action and/or may themselves represent potential drug targets. Investigation of the mechanisms of action of these agents may lead to the design of potent and selective antimalarial and other antiparasitic drugs. This review discusses the properties of immunophilins in parasites and the 'animal model' <i>Caenorhabditis elegans</i> and relates these to our understanding of the roles of these proteins in cellular biochemistry, host-parasite interaction and the antiparasitic mechanisms of the drugs that bind to them

Genotype-phenotype analysis of von Hippel-Lindau syndrome in Korean families: HIF-α binding site missense mutations elevate age-specific risk for CNS hemangioblastoma

BACKGROUND: von Hippel-Lindau (VHL) disease is a rare hereditary tumor syndrome caused by VHL gene mutations that is characterized by heterogeneous phenotypes such as benign/malignant tumors of the central nervous system, retina, kidney, adrenal gland, and pancreas. The genotype-phenotype correlation has not been well characterized in the Korean population so far. Therefore, this study aimed to evaluate the VHL mutation spectrum and genotype-phenotype correlations in Korean VHL patients. METHODS: Thirteen unrelated subjects with VHL mutations were included. Direct sequencing and multiplex ligation-dependent probe amplification were performed. Consequently, the clinical manifestations and family histories of the subjects were evaluated. RESULTS: We identified 10 different VHL mutations. The c.160_161delAT frameshift mutation was novel. Missense mutations clustered in 2 domains (α domain in exon 1; β domain in exon 3). The most frequently observed mutation was c.208G > A (p.Glu70Lys). Milder phenotypes were observed in subjects with de novo mutations. Age-specific risk for CNS hemangioblastoma was significantly higher in subjects carrying missense mutations within the HIF-α binding site (P < 0.05). CONCLUSIONS: This study provides insight into the genotype-phenotype correlation in that amino acid substitutions in the HIF-α binding site may predispose patients to age-related risks of CNS hemangioblastoma

Use of Zebrafish to Probe the Divergent Virulence Potentials and Toxin Requirements of Extraintestinal Pathogenic Escherichia coli

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms

Phosphorylation of Mcm2 modulates Mcm2–7 activity and affects the cell’s response to DNA damage

The S-phase kinase, DDK controls DNA replication through phosphorylation of the replicative helicase, Mcm2–7. We show that phosphorylation of Mcm2 at S164 and S170 is not essential for viability. However, the relevance of Mcm2 phosphorylation is demonstrated by the sensitivity of a strain containing alanine at these positions (mcm2AA) to methyl methanesulfonate (MMS) and caffeine. Consistent with a role for Mcm2 phosphorylation in response to DNA damage, the mcm2AA strain accumulates more RPA foci than wild type. An allele with the phosphomimetic mutations S164E and S170E (mcm2EE) suppresses the MMS and caffeine sensitivity caused by deficiencies in DDK function. In vitro, phosphorylation of Mcm2 or Mcm2EE reduces the helicase activity of Mcm2–7 while increasing DNA binding. The reduced helicase activity likely results from the increased DNA binding since relaxing DNA binding with salt restores helicase activity. The finding that the ATP site mutant mcm2K549R has higher DNA binding and less ATPase than mcm2EE, but like mcm2AA results in drug sensitivity, supports a model whereby a specific range of Mcm2–7 activity is required in response to MMS and caffeine. We propose that phosphorylation of Mcm2 fine-tunes the activity of Mcm2–7, which in turn modulates DNA replication in response to DNA damage

Maternal Dietary Supplementation with Oligofructose-Enriched Inulin in Gestating/Lactating Rats Preserves Maternal Bone and Improves Bone Microarchitecture in Their Offspring

This study received financial support from Abbott Nutrition, a commercial company, and coauthors PBV, MM, JMLP and RR are employees of Abbott Nutrition. There are two patents related with the data presented (EP 2502507 A1 and EP 2745706 A1).Some of these results were presented in the 7th World Congress of DOHaD (2011) and in the World Congress on Osteoporosis, Osteoarthritis and Musculoskeletal Disease (WCO-IOF-ESCEO) (2014).Nutrition during pregnancy and lactation could exert a key role not only on maternal bone, but also could influence the skeletal development of the offspring. This study was performed in rats to assess the relationship between maternal dietary intake of prebiotic oligofructose-enriched inulin and its role in bone turnover during gestation and lactation, as well as its effect on offspring peak bone mass/architecture during early adulthood. Rat dams were fed either with standard rodent diet (CC group), calcium-fortified diet (Ca group), or prebiotic oligofructose-enriched inulin supplemented diet (Pre group), during the second half of gestation and lactation. Bone mineral density (BMD) and content (BMC), as well as micro-structure of dams and offspring at different stages were analysed. Dams in the Pre group had significantly higher trabecular thickness (Tb.Th), trabecular bone volume fraction (BV/TV) and smaller specific bone surface (BS/BV) of the tibia in comparison with CC dams. The Pre group offspring during early adulthood had an increase of the lumbar vertebra BMD when compared with offspring of CC and Ca groups. The Pre group offspring also showed significant increase versus CC in cancellous and cortical structural parameters of the lumbar vertebra 4 such as Tb.Th, cortical BMD and decreased BS/BV. The results indicate that oligofructose-enriched inulin supplementation can be considered as a plausible nutritional option for protecting against maternal bone loss during gestation and lactation preventing bone fragility and for optimizing peak bone mass and architecture of the offspring in order to increase bone strength.This study was funded by Abbott Nutrition R&D, and co-authors PBV, MM, JMLP and RR receive salary from Abbott Nutrition

PathogenFinder - Distinguishing Friend from Foe Using Bacterial Whole Genome Sequence Data.

Although the majority of bacteria are harmless or even beneficial to their host, others are highly virulent and can cause serious diseases, and even death. Due to the constantly decreasing cost of high-throughput sequencing there are now many completely sequenced genomes available from both human pathogenic and innocuous strains. The data can be used to identify gene families that correlate with pathogenicity and to develop tools to predict the pathogenicity of newly sequenced strains, investigations that previously were mainly done by means of more expensive and time consuming experimental approaches. We describe PathogenFinde

Gata4 Is Required for Formation of the Genital Ridge in Mice

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonadHoward Hughes Medical Institut

Multiway modeling and analysis in stem cell systems biology

<p>Abstract</p> <p>Background</p> <p>Systems biology refers to multidisciplinary approaches designed to uncover emergent properties of biological systems. Stem cells are an attractive target for this analysis, due to their broad therapeutic potential. A central theme of systems biology is the use of computational modeling to reconstruct complex systems from a wealth of reductionist, molecular data (e.g., gene/protein expression, signal transduction activity, metabolic activity, etc.). A number of deterministic, probabilistic, and statistical learning models are used to understand sophisticated cellular behaviors such as protein expression during cellular differentiation and the activity of signaling networks. However, many of these models are bimodal i.e., they only consider row-column relationships. In contrast, multiway modeling techniques (also known as tensor models) can analyze multimodal data, which capture much more information about complex behaviors such as cell differentiation. In particular, tensors can be very powerful tools for modeling the dynamic activity of biological networks over time. Here, we review the application of systems biology to stem cells and illustrate application of tensor analysis to model collagen-induced osteogenic differentiation of human mesenchymal stem cells.</p> <p>Results</p> <p>We applied Tucker1, Tucker3, and Parallel Factor Analysis (PARAFAC) models to identify protein/gene expression patterns during extracellular matrix-induced osteogenic differentiation of human mesenchymal stem cells. In one case, we organized our data into a tensor of type protein/gene locus link × gene ontology category × osteogenic stimulant, and found that our cells expressed two distinct, stimulus-dependent sets of functionally related genes as they underwent osteogenic differentiation. In a second case, we organized DNA microarray data in a three-way tensor of gene IDs × osteogenic stimulus × replicates, and found that application of tensile strain to a collagen I substrate accelerated the osteogenic differentiation induced by a static collagen I substrate.</p> <p>Conclusion</p> <p>Our results suggest gene- and protein-level models whereby stem cells undergo transdifferentiation to osteoblasts, and lay the foundation for mechanistic, hypothesis-driven studies. Our analysis methods are applicable to a wide range of stem cell differentiation models.</p