Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Congenic Mapping and Allele-Specific Alteration Analysis of Stmm1 Locus Conferring Resistance to Early-Stage Chemically Induced Skin Papillomas

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

Effects of Regulator of G Protein Signaling 19 (RGS19) on Heart Development and Function

Wnt/Wg genes play a critical role in the development of various organisms. For example, the Wnt/β-catenin signal promotes heart formation and cardiomyocyte differentiation in mice. Previous studies have shown that RGS19 (regulator of G protein signaling 19), which has Gα subunits with GTPase activity, inhibits the Wnt/β-catenin signal through inactivation of Gα_o. In the present study, the effects of RGS19 on mouse cardiac development were observed. In P19 teratocarcinoma cells with RGS19 overexpression, RGS19 inhibited cardiomyocyte differentiation by blocking the Wnt signal. Additionally, several genes targeted by Wnt were down-regulated. For the in vivo study, we generated RGS19-overexpressing transgenic (RGS19 TG) mice. In these transgenic mice, septal defects and thin-walled ventricles were observed during the embryonic phase of development, and the expression of cardiogenesis-related genes, BMP4 and Mef2C, was reduced significantly. RGS19 TG mice showed increased expression levels of brain natriuretic peptide and β-MHC, which are markers of heart failure, increase of cell proliferation, and electrocardiogram analysis shows abnormal ventricle repolarization. These data provide in vitro and in vivo evidence that RGS19 influenced cardiac development and had negative effects on heart function.     

Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11

Background

Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody.

Methodology/Principal Findings

We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 µm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11.

Conclusions/Significance

Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host.     

CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca²⁺ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca²⁺-regulated proteins that—in mature sperm—are involved in flagellar bending.     

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.     

A Novel Two-Component Signaling System Facilitates Uropathogenic Escherichia coli's Ability to Exploit Abundant Host Metabolites

Two-component signaling systems (TCSs) are major mechanisms by which bacteria adapt to environmental conditions. It follows then that TCSs would play important roles in the adaptation of pathogenic bacteria to host environments. However, no pathogen-associated TCS has been identified in uropathogenic Escherichia coli (UPEC). Here, we identified a novel TCS, which we termed KguS/KguR (KguS: α-ketoglutarate utilization sensor; KguR: α-ketoglutarate utilization regulator) in UPEC CFT073, a strain isolated from human pyelonephritis. kguS/kguR was strongly associated with UPEC but was found only rarely among other E. coli including commensal and intestinal pathogenic strains. An in vivo competition assay in a mouse UTI model showed that deletion of kguS/kguR in UPEC CFT073 resulted in a significant reduction in its colonization of the bladders and kidneys of mice, suggesting that KguS/KguR contributed to UPEC fitness in vivo. Comparative proteomics identified the target gene products of KguS/KguR, and sequence analysis showed that TCS KguS/KguR and its targeted-genes, c5032 to c5039, are encoded on a genomic island, which is not present in intestinal pathogenic E. coli. Expression of the target genes was induced by α-ketoglutarate (α-KG). These genes were further shown to be involved in utilization of α-KG as a sole carbon source under anaerobic conditions. KguS/KguR contributed to the regulation of the target genes with the direct regulation by KguR verified using an electrophoretic mobility shift assay. In addition, oxygen deficiency positively modulated expression of kguS/kguR and its target genes. Taken altogether, this study describes the first UPEC-associated TCS that functions in controlling the utilization of α-ketoglutarate in vivo thereby facilitating UPEC adaptation to life inside the urinary tract.     

Secreted Frizzled-related Protein 1 (Sfrp1) Regulates the Progression of Renal Fibrosis in a Mouse Model of Obstructive Nephropathy

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1^−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1^−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.     

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

Schistosoma mansoni venom allergen like proteins present differential allergic responses in a murine model of airway inflammation

Background

The Schistosoma mansoni Venom-Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in the host-pathogen interaction. Some of these molecules were suggested by us and others as potential immunomodulators and vaccine candidates, due to their functional classification, expression profile and predicted localization. From a vaccine perspective, one of the concerns is the potential allergic effect of these molecules.

Methodology/Principal Findings

Herein, we characterized the putative secreted proteins SmVAL4 and SmVAL26 and explored the mouse model of airway inflammation to investigate their potential allergenic properties. The respective recombinant proteins were obtained in the Pichia pastoris system and the purified proteins used to produce specific antibodies. SmVAL4 protein was revealed to be present only in the cercarial stage, increasing from 0–6 h in the secretions of newly transformed schistosomulum. SmVAL26 was identified only in the egg stage, mainly in the hatched eggs'' fluid and also in the secretions of cultured eggs. Concerning the investigation of the allergic properties of these proteins in the mouse model of airway inflammation, SmVAL4 induced a significant increase in total cells in the bronchoalveolar lavage fluid, mostly due to an increase in eosinophils and macrophages, which correlated with increases in IgG1, IgE and IL-5, characterizing a typical allergic airway inflammation response. High titers of anaphylactic IgG1 were revealed by the Passive Cutaneous Anaphylactic (PCA) hypersensitivity assay. Additionally, in a more conventional protocol of immunization for vaccine trials, rSmVAL4 still induced high levels of IgG1 and IgE.

Conclusions

Our results suggest that members of the SmVAL family do present allergic properties; however, this varies significantly and therefore should be considered in the design of a schistosomiasis vaccine. Additionally, the murine model of airway inflammation proved to be useful in the investigation of allergic properties of potential vaccine candidates.     

Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.