P42 Ebp1 functions as a tumor suppressor in non-small cell lung cancer

Although the short isoform of ErbB3-binding protein 1 (Ebp1), p42 has been considered to be a potent tumor suppressor in a number of human cancers, whether p42 suppresses tumorigenesis of lung cancer cells has never been clarified. In the current study we investigated the tumor suppressor role of p42 in non-small cell lung cancer cells. Our data suggest that the expression level of p42 is inversely correlated with the cancerous properties of NSCLC cells and that ectopic expression of p42 is sufficient to inhibit cell proliferation, anchorage-independent growth, and invasion as well as tumor growth in vivo. Interestingly, p42 suppresses Akt activation and overexpression of a constitutively active form of Akt restores the tumorigenic activity of A549 cells that is ablated by exogenous p42 expression. Thus, we propose that p42 Ebp1 functions as a potent tumor suppressor of NSCLC through interruption of Akt signaling. [BMB Reports 2015; 48(3): 159-165]     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain

ABSTRACT: BACKGROUND: The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area endemic with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes. RESULTS: Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas. CONCLUSIONS: The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.     

Distribution and morphology of ghrelin immunostained cells in the adrenal gland of the African ostrich

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor. We investigated the distribution and morphological characteristics of ghrelin-immunopositive (ghrelin-ip) cells in the African ostrich adrenal gland. We found that the adrenal gland of the African ostrich consisted of three parts: capsule, inter-renal tissue and chromaffin cells. The inter-renal tissue and chromaffin cells interdigitated irregularly. The inter-renal tissue consisted of a peripheral zone and a central inner zone. The peripheral zone could be divided into an outer subcapsular zone and an inner zone. The subcapsular zone cells were arranged as a bow, while the inner area cells formed cords that were perpendicular to the capsule. The central inner zone exhibited irregular clumps and the cells were morphologically similar to chromaffin cells. Ghrelin-ip cells were located throughout the adrenal gland except the capsule. The majority of ghrelin-ip cells were found among the chromaffin cells. The number of ghrelin-ip cells in the inter-renal tissue decreased gradually from the central inner zone, to the inner zone to the subcapsular zone. The ghrelin-ip cells were oval or irregular in shape and exhibited cytoplasmic staining. Our findings suggest that ghrelin may play a role in regulating adrenal hormone secretion in the African ostrich.     

ICEA of Mycoplasma agalactiae: a new family of self‐transmissible integrative elements that confers conjugative properties to the recipient strain

Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self‐transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE‐negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer‐deficient mutants indicated that this process requires an ICEA‐encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA‐encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far‐reaching implications given their minute‐size genome and the number of species that are pathogenic for a broad host‐range.     

Emulsifying properties of biodegradable polylactide-grafted dextran copolymers

Amphiphilic glycopolymers, polylactide-grafted dextran copolymers (Dex-g-PLA), were synthesized with a well-controlled architecture obtained through a three-step procedure: partial silylation of the dextran hydroxyl groups, ring-opening polymerization of D,L-lactide initiated from remaining hydroxyl groups, silylether deprotection under very mild conditions. Depending on their proportion in polylactide (PLA), these copolymers exhibited solubility either in water or in organic solvents. The emulsifying properties of these glycopolymers were studied: depending on their PLA-to-dextran ratio, they were able to stabilize either direct or inverse emulsions. Droplet size was related to the amount of amphiphilic copolymer in the continuous phase. The aging mechanism of both direct and inverse emulsions was shown to be Ostwald ripening in the first weeks following preparation. Finally inverse miniemulsion copolymerization of acrylamide and N, N'-methylenebisacrylamide was performed in the presence of an amphiphilic Dex-g-PLA stabilizer. Polyacrylamide hydrogel nanoparticles were prepared in that way.     

Occurrence,Plasticity, and Evolution of the vpma Gene Family,a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.Bacteria of the Mycoplasma genus belong to the class Mollicutes and represent a remarkable group of organisms that derived from the Firmicutes lineage by massive genome reduction (41, 51). Consequent to this regressive evolution, modern mycoplasmas have been left with small genomes (580 to 1,400 kb), a limited number of metabolic pathways, and no cell wall. Due to these particularities, members of the Mycoplasma genus have often been portrayed as “minimal self-replicating organisms.” Despite this apparent simplicity, a large number of mycoplasma species are successful pathogens of humans and a wide range of animals, in which they are known to cause diseases that are often chronic and debilitating (1, 33). The surface of their single membrane is considered a key interface in mediating adaptation and survival in the context of a complex, immunocompetent host (10, 13, 34, 40). Indeed, mycoplasmas possess a highly versatile surface architecture due to a number of sophisticated genetic systems that promote intraclonal variation in the expression and structure of abundant surface lipoproteins (9, 50). Usually, these systems combine a set of contingency genes with a molecular switch for turning expression on or off that is based on either (i) spontaneous mutation (slipped-strand mispairing), (ii) gene conversion, or (iii) specific DNA rearrangements (9). While high-frequency phenotypic variation using the two first mechanisms has been described thoroughly for other bacteria (47), switching of surface components by shuffling of silent genes at a particular single expression locus has been studied mainly in mycoplasmas (3, 8, 14, 16, 23, 39, 43).Mycoplasma agalactiae, an important pathogen responsible for contagious agalactia in small ruminants (listed by the World Organisation for Animal Health), possesses a family of lipoproteins encoded by the vpma genes for which phase variation in expression is driven by a “cut-and-paste” mechanism involving a tyrosine site-specific recombinase designated Xer1 (16). Data previously gathered with the PG2 type strain identified a single vpma cluster (42) composed of six vpma genes adjacent to one xer1 gene (Fig. ​(Fig.1A).1A). Based on fine genetic analyses, Xer1 was further shown to mediate frequent site-specific DNA rearrangements by targeting short DNA sequences located upstream of each vpma gene (8, 16). While some vpma rearrangements can be phenotypically silent, others result in Vpma on-off switching by linking a silent vpma gene sequence immediately downstream of the unique vpma promoter. Because site-specific recombination can be reciprocal, the initial vpma configuration can be restored without a loss of genetic information.Open in a separate windowFIG. 1.Comparison of M. agalactiae vpma loci between the PG2 type strain and strain 5632. Schematics represent the organization of the vpma loci in clonal variant 55.5 derived from PG2 (16, 42) (A) and in clonal variant c1 derived from strain 5632 (B). (C) Counterpart of locus II₅₆₃₂ in PG2 showing the absence of vpma genes in this region. (D) The presence of two distinct loci in 5632 was confirmed by PCR, using the primer pair xerF-phydR or xerF-agpR, and the resulting amplicons are shown. The locations of the primers are indicated by arrowheads in panels A, B, and C. Large white arrows labeled with letters represent Vpma CDSs. The positions of the promoters are represented by black arrowheads labeled “P.” The two non-Vpma-related CDSs (abiG_I and abiG_II) are indicated by large arrows filled with a dotted pattern. ISMag1 elements are indicated by hatched boxes. Recombination sites downstream of each vpma gene are indicated by black dots. An asterisk indicates that the corresponding vpma gene is present at two distinct loci. Schematics were drawn approximately to scale. HP, hypothetical protein; CHP, conserved hypothetical protein. Small letters and bars indicate the positions of short particular sequences mentioned in the text and in Fig. ​Fig.33 and ​and4.4. The pictures on the left side of panels A and B illustrate the variable surface expression of Vpma, as previously described (8, 17). These correspond to colony immunoblots using Vpma-specific polyclonal antibodies recognizing PG2 VpmaW (α W) and VpmaY (α Y) epitopes.The vsa family of the murine pathogen M. pulmonis (3, 39), the vsp family of the bovine pathogen M. bovis (2, 24), and the vpma family of M. agalactiae all generate intraclonal surface diversity by using very similar molecular switches (23), although their overall coding sequences seem to be specific to the Mycoplasma species. DNA rearrangements also govern phase variation of the 38 mpl genes of the human pathogen M. penetrans (27, 35, 38). However, in this mycoplasma species the molecular switch is slightly different, since each mpl gene possesses its own invertible promoter (19). In M. penetrans, the individual expression of each mpl gene can then be switched on and off in a combinatory manner, resulting in a large number of possible Mpl surface configurations. Since M. pulmonis, M. bovis, and M. agalactiae all belong to the Mycoplasma hominis phylogenetic cluster (48) and are relatively closely related, while M. penetrans belongs to the distinct Mycoplasma pneumoniae phylogenetic cluster (30, 48), it is tempting to speculate that the vsa, vsp, and vpma systems were all inherited from a common ancestor and that the bulk of their coding sequences evolved independently in their respective hosts while the molecular switch mechanism was retained.In so-called “minimal” bacteria, the occurrence of relatively large genomic portions dedicated to multigene families, with genes encoding phase-variable, related surface proteins, suggests that they serve an important function(s). Data accumulated over the years for several mycoplasma species tend to indicate that one general purpose of these systems is to provide the mycoplasma with a variable shield that modulates surface accessibility in order to escape the host response and to adapt to rapidly changing environments (10, 11, 13, 40, 50). On the other hand, the sequences of phase-variable proteins are relatively conserved within one species but divergent between species, suggesting a more specific role for these molecules.The role of the Vpma family of M. agalactiae has yet to be elucidated, but it was recently shown that Vpma switches in expression occur at a remarkably high rate in vitro (10⁻² to 10⁻³ per cell per generation) (8, 17). The vpma systems described for PG2 (16) and another M. agalactiae strain, isolated in Israel (in which Vpmas were designated Avg proteins [14]), both revealed a repertoire of six vpma genes and only one promoter, suggesting that in M. agalactiae the number of Vpma configurations is limited to six. This contrasts with the situation commonly found in other Mycoplasma variable systems, which can offer a larger mosaic of surface architecture because of the concomitant switches of several related surface proteins and/or because of a larger number of phase-variable genes.To further understand the degree of diversity that can be generated at the surface of M. agalactiae, we analyzed the vpma gene content of a field strain, 5632, whose genome was recently sequenced by our group (unpublished data). The present study shows that 5632 contains a total of 23 vpma genes distributed in two distinct loci that both contain a recombinase gene. Further genomic and proteomic analyses indicated that the capacity of 5632 to vary its Vpma surface architecture is far more complex than that described for the type strain. Unlike the case for PG2, both 5632 vpma loci are associated with several mobile genetic insertion elements (IS) that could play an evolutionary role in the dynamics of vpma repertoires, as suggested by data presented here. One 5632 vpma locus contains open reading frames (ORFs) that are highly conserved in both M. bovis, a closely related bovine mycoplasma, and the phylogenetically distant mycoplasmas of the M. mycoides cluster, which are also important ruminant pathogens. Whether these were acquired through evolution or through horizontal transfer is discussed. The present study reveals an additional degree of complexity for the Vpma system and further suggests that some field strains might have more dynamic genomes and a more variable surface than was first estimated (42).