Glycoproteomic survey of Mammillaria gracillis tissues grown in vitro

The structure elucidation of protein-linked N-glycans in plants has raised interest in the past years due to remarkable physiological roles attributed to these modifications. However, little information about the glycoprotein patterns related to plant cell differentiation, dedifferentiation and transformation is available. In this work, the use of two-dimensional polyacrylamide gel electrophoresis in conjunction with matrix assisted laser/desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the characterization of carbohydrates released from plant glycoproteins is described. Proteins from different Mammillaria tissues (shoot, callus, hyperhydric regenerant, and TW tumor) were separated by 2D SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and incubated with Con A to detect N-glycosylated proteins. To discover if the same protein can have various N-glycan structures depending on the organization status of the tissue, the selected glycoprotein spot, which was common for all investigated tissues, was excised from the gels and digested by PNGase A. The released oligosaccharides were analyzed by MALDI-TOF-MS. The results obtained in this study indicate that the N-glycosylation pattern of the protein is clearly dependent on level of plant tissue organization and can be related to the specific morphogenic status.     

Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.     

DNA marker-assisted evaluation of fruit firmness at harvest and post-harvest fruit softening in a diverse apple germplasm

Several different genes have been proposed as responsible for fruit texture variability at harvest and/or after storage. We have analysed 127 apple cultivars for allelic composition in two key genes that are directly involved in the ethylene biosynthesis pathway, Md-ACS1 and Md-ACO1, and two other genes that are involved in cell wall degradation, Md-Exp7 and Md-PG1. Firmness was measured with a penetrometer at harvest and after 6 or 12 weeks (early- and late-maturing cultivars, respectively) of cold storage. Maturation time was positively correlated with firmness at harvest and negatively correlated with fruit softening rate (difference between firmness at harvest and after storage, divided by number of weeks in storage). Polyploid cultivars showed significantly higher firmness at harvest compared to diploids. Alleles previously described as responsible for good texture were associated with significantly lower softening for Md-ACS1 and Md-PG1, but the opposite was noted for Md-EXP7. Results were nonsignificant for Md-ACO1. Allele frequencies were very uneven in all four loci, with the three most common multi-locus configurations accounting for 64 % of the entire material. The predictive power of these genes was calculated with a partial least squares discriminant analysis, and these accounted for 15 % of the observed variation in initial firmness and 18 % for softening rate. Inclusion of maturation time, storage time (i.e. 6 or 12 weeks) and initial firmness into the model however increased the predictability of softening rate to 38 %. Dividing the material in modern (released after 1960) and old cultivars did not change the outcome of our analyses.     

FRAX597, a Small Molecule Inhibitor of the p21-activated Kinases,Inhibits Tumorigenesis of Neurofibromatosis Type 2 (NF2)-associated Schwannomas

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.     

Sex Determination in 58 Bird Species and Evaluation of CHD Gene as a Universal Molecular Marker in Bird Sexing

The aim of this research was to test the CHD gene (Chromo Helicase DNA‐binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim. Zoo Biol 32:269–276, 2013. © 2012 Wiley Periodicals, Inc.     

Substrate positioning in chitinase A, a processive chito-biohydrolase from Serratia marcescens

The contributions of the -3 subsite and a putative +3 subsite to substrate positioning in ChiA from Serratia marcescens have been investigated by comparing how ChiA and its -3 subsite mutant W167A interact with soluble substrates. The data show that Trp - GlcNAc stacking in the -3 subsite rigidifies the protein backbone supporting the formation of the intermolecular interaction network that is necessary for the recognition and positioning of the N-acetyl groups before the -1 subsite. The +3 subsite exhibits considerable substrate affinity that may promote endo-activity in ChiA and/or assist in expelling dimeric products from the +1 and +2 subsites during processive hydrolysis.     

Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".     

Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.     

Synthesis, cytostatic activity and ADME properties of C-5 substituted and N-acyclic pyrimidine derivatives

The synthesis of the novel 5-alkyl pyrimidine derivatives, 5,6-dihydrofuro[2,3-d]pyrimidines and 5-alkyl N-methoxymethyl pyrimidine derivatives and evaluation of their cytostatic activities are described. The mechanism of antiproliferative effect of 5-(2-chloroethyl)-substituted pyrimidine 3 that exerted the pronounced cytostatic activity was studied in further details on colon carcinoma (HCT116) cells. The cell cycle perturbation analysis demonstrated severe DNA damage (G2/M arrest) pointing to a potential DNA alkylating ability of 3. Preliminary ADME data of 3 and its 6-methylated structural congener (6-Me-3) showed their high permeability and good metabolic stability.     

Microscopic anatomy of male tegumental glands and associated cuticular structures in Titanethes albus (Crustacea: Isopoda)

Male glandular organs characterized by porous surfaces with hair-like cuticular elaborations are known from several trichoniscid isopods. In the subterranean species Titanethes albus, males possess paired tubercles with numerous hairs and pores dorsally on the pleon. We analyzed the microscopic anatomy of these structures with scanning and transmission electron microscopy. Diverse epicuticular formations and numerous sensilla, which are probably chemoreceptive, are present on the tubercles. We found several secretory surfaces on the pleon in addition to the dorsal tubercles. We also examined the distribution, architecture and ultrastructure of male-specific glands in T. albus with light and transmission electron microscopy. Three distinct types of male-specific rosette glands are present in different parts of the pleon and in the uropods. Glands secreting on the dorsal tubercles contain stellar central cells. The ultrastructure and histochemical staining properties of male-specific glands in T. albus suggest that they produce peptides which might function as contact pheromones.