Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B

Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.     

AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.     

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.     

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.Bacterial lipoproteins (Lpps)¹ are a subset of membrane proteins that are covalently modified with a lipidic moiety at their N-terminal cysteine residue. It is commonly reported that Lpps of Gram-positive bacteria are processed by two key enzymes; the prolipoprotein diacylglyceryl transferase (Lgt) and the lipoprotein signal peptidase (Lsp). The Lgt enzyme recognizes a so-called lipobox motif in the C-terminal region of the signal peptide of a premature lipoprotein and transfers a diacylglyceryl moiety to the cysteine residue of the lipobox (1), (2). Subsequently, the Lsp enzyme cleaves the signal peptide resulting in a mature Lpp (3), (4). Nevertheless, recent reports have suggested that N-acylation occurs in bacteria that lack the Gram-negative homologous apolipoprotein N-acyltransferase (Lnt) gene responsible for this modification (5, 6), and that Lpp N-terminal could also be modified with an acetyl group in some Gram-positive (7).Lpps have been described as virulence factors because they play critical roles in membrane stabilization, nutrient uptake, antibiotic resistance, bacterial adhesion to host cells, protein maturation and secretion and many of them still have unknown function (8). Several studies have suggested that bacterial Lpps are pathogen-associated molecular patterns (PAMPs) sensed by the mammalian host through Toll-like receptor 2 (TLR2) heterodimerized with TLR1 or TLR6 to induce innate immunity activation and to control adaptive immunity (9–12). TLR2 plays a critical role in the host response to the Gram-positive bacteria Staphylococcus aureus (13) and Streptococcus agalactiae (14). Although TLR2 has been considered a receptor for various structurally unrelated PAMPs, recent studies have suggested that, via their lipid moiety, bacterial Lpps function as the major, if not the sole, ligand molecules responsible for TLR2 activation (15). Although Gram-negative Lpps have been widely studied, little information is available for Gram-positive Lpps (16) and the ways they are released into the bacterial extracellular compartment and reach the host immune system remain unclear.We focused our attention on Lpps release by Streptococcus pyogenes. This Gram-positive bacterium is an important human pathogen that causes a wide range of diseases from superficial and self-limiting infection, e.g. pharyngitis and impetigo, to more systemic or invasive diseases like necrotizing fasciitis and septicemia (17). Understanding the role of bacterial Lpps in mediating innate and acquired immunity can be instrumental for the therapy and prophylaxis of human S. pyogenes infections. In this study, we showed that in S. pyogenes Lpps are released into the growth medium within vesicle-like structures in minute amounts. Conditions weakening the bacterial cell wall, such as the addition of sublethal concentrations of penicillin to the bacterial growth medium enhanced this phenomenon and allowed the recovery of sufficient material to enable an in-depth characterization. Proteomic analysis of the vesicles revealed that they were almost exclusively constituted of Lpps. A total of 28 Lpps were identified, representing more than 72% of the Lpps predicted from the genome of the strain under investigation. In addition, multiple transmembrane domain proteins were not found in abundance associated to the vesicles, indicating that vesicles were not representative of the bacterial membrane. We defined these vesicles as Lipoprotein-rich Membrane Vesicles (LMVs).Common characteristics are shared between the LMVs and the ExPortal described for the first time by Rosch and Caparon (18). This asymmetric and distinct membrane microdomain has been reported to be enriched in anionic phospholipids and acts in promoting the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and the accessory factors required for their maturation (19–21). An association between ExPortal and peptidoglycan synthesis has also been reported (22). Similarly, LMVs are enriched in anionic phosphatidylglycerol, enzymes involved in protein maturation/secretion and cell wall biogenesis, suggesting that LMVs might derive from the ExPortal. Finally, we showed that LMVs do not induce TLR2 activation, indicating that the Lpps did not act as PAMPs when integrated into the LMVs.     

The fatty acid profiles in a drop of blood from a fingertip correlate with physiological, dietary and lifestyle parameters in volunteers

Limited data are available on the fatty acid (FA) composition of circulating lipids and the associations with diet, physiological and pathological conditions, due to the complexity and costs of the analytical process. The aim of our study was to evaluate the FA composition in 108 healthy subjects and to correlate the data with gender, pregnancy, dietary habits, lifestyle, and short-term controlled intake of n-3 FA, using an innovative analytical approach for the collection and processing of blood samples. Ten subjects were also supplemented with n-3 polyunsaturated FA as smoked salmon or capsules for 3 weeks. The resulting blood FA composition was affected by gender, pregnancy, diet and smoking. The data indicate that this new analytical methodology is suitable for assessing associations between circulating FA and various parameters in large population groups, and is applicable to epidemiological studies and in the assessment of the effects of controlled FA supplementation in clinical studies.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

AQP1 expression analysis in human diseases: implications for proteomic characterization

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.