A 43 kDa protein inserted at the plasma membrane-cytoskeleton interface in rumen entodiniomorphid ciliates

Summary The P-43 ofEudiplodinium and homologous proteins in three other entodiniomorphid species, free-living ciliates, flagellates, and HeLa cells, were identified at the plasma membrane-cytoskeleton interface. Proteins cross-reacting with MAb B6 were also located at the ciliary inner surface of the plasma membrane. Due to the strong adhesion of the plasma membrane to the underlying cytoskeleton, classical extraction with detergents, urea, NaOH, and PTA, failed to separate the two components completely. However, the extraction properties of P-43, associated with its membrane-cytoskeleton interactive functions, suggest that this unglycosylated protein may present some analogies with proteins of the intermediate filaments. Their ubiquity and localization suggest that P-43 and MAb B6 crossreacting proteins may not be strictly epiplasmic but could be amphitropic proteins, strongly anchored to both the plasma membrane and the underlying microfilament framework, via protein-protein binding or by direct insertion in the lipid bilayer.Abbreviations BSA bovine serum albumin - Con A concanavalin A - EDTA ethylene diamine tetraacetic acid - EM electron microscopy - IF intermediate filaments - MAb monoclonal antibody - MET 2-mercaptoethanol - MW molecular weight - PAb polyclonal antibody - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PTA phosphotungstic acid - SDS sodiumdodecyl sulfate - TAME Na-p-tosyl-arginine methyl ester - TLCK Na-p-tosyl-lysine chloromethyl ketone     

Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12),a key enzyme ofcarbon metabolism,was purified and characterized to homogeneity from skeletal muscle of Camelusdromedarius.The protein was purified approximately 26.8 folds by conventional ammonium sulphatefractionation followed by Blue Sepharose CL-6B chromatography,and its physical and kinetic propertieswere investigated.The native protein is a homotetramer with an apparent molecular weight of approximately146 kDa.Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectricpoint of 7.2.The optimum pH of the purified enzyme was 7.8.Studies on the effect of temperature onenzyme activity revealed an optimal value of approximately 28-32 ℃ with activation energy of 4.9 kcal/mol.The apparent K_m values for NAD~ and DL-glyceraldehyde-3-phophate were estimated to be 0.025±0.040mM and 0.21±0.08 mM, respectively. The V_(max) of the purified protein was estimated to be 52.7±5.9 U/mg.These kinetic parameter values were different from those described previously, reflecting protein differencesbetween species.     

Evolutionary relationships among heterokont algae (the autotrophic stramenopiles) based on combined analyses of small and large subunit ribosomal RNA

In order to study the phylogenetic relationships within the stramenopiles, and particularly among the heterokont algae, we have determined complete or nearly complete large-subunit ribosomal RNA sequences for different species of raphidophytes, phaeophytes, xanthophytes, chrysophytes, synurophytes and pinguiophytes. With the small- and large-subunit ribosomal RNA sequences of representatives for nearly all known groups of heterokont algae, phylogenetic trees were constructed from a concatenated alignment of both ribosomal RNAs, including more than 5,000 positions. By using different tree construction methods, inferred phylogenies showed phaeophytes and xanthophytes as sister taxa, as well as the pelagophytes and dictyochophytes, and the chrysophytes/synurophytes and eustigmatophytes. All these relationships are highly supported by bootstrap analysis. However, apart from these sister group relationships, very few other internodes are well resolved and most groups of heterokont algae seem to have diverged within a relatively short time frame.     

Decorated surfaces by biofunctionalized gold beads: application to cell adhesion studies

We describe a simple but versatile method to decorate solid surfaces randomly with colloidal gold particles to which ligands of cell receptors can be coupled to generate local attraction sites for the control of cell adhesion. A self-assembled monolayer of (3-mercaptopropyl)trimethoxysilane was deposited on glass slides. Gold beads were anchored to the functionalized surface through the sulfur group. We characterized the gold bead distribution on the functionalized surface with reflection interference contrast microscopy. The gold beads were functionalized with a disulfide-coupled cyclic pentapeptide containing an arginine-glycine-aspartic acid (RGD) tripeptide sequence which is selectively recognized by integrin receptors alpha(V)beta(3) of endothelial cells. A blocking layer of bovine serum albumin was adsorbed onto the surface to prevent non-specific binding of the cells. We demonstrate that the RGD-functionalized colloidal gold beads act as local attraction centers, mediating rapid cell anchoring on a substrate impeding cell adhesion in the absence of attraction centers. Surprisingly, microinterferometry shows that after a time delay of about 1 h, the regions of the cell surface between the gold beads form close contacts with the substrate, which is attributed to strong van der Waals attraction after escape of repeller molecules from the contact surface.     

Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomyces: Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.     

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.     

A new method for the extraction of fetal ECG from the dependent abdominal signals using blind source separation and adaptive noise cancellation techniques

Background

The electrocardiogram (ECG) is a diagnostic tool that records the electrical activity of the heart, and depicts it as a series of graph-like tracings, or waves. Being able to interpret these details allows diagnosis of a wide range of heart problems. Fetal electrocardiogram (FECG) extraction has an important impact in medical diagnostics during the mother pregnancy period. Since the observed FECG signals are often mixed with the maternal ECG (MECG) and the noise induced by the movement of electrodes or by mother motion, the separation process of the ECG signal sources from the observed data becomes quite complicated. One of its complexity is when the ECG sources are dependent, thus, in this paper we introduce a new approach of blind source separation (BSS) in the noisy context for both independent and dependent ECG signal source. This approach consist in denoising the observed ECG signals using a bilateral total variation (BTV) filter; then minimizing the Kullbak-Leibler divergence between copula densities to separate the FECG signal from the MECG one.

Results

We present simulation results illustrating the performance of our proposed method. We will consider many examples of independent/dependent source component signals. The results will be compared with those of the classical method called independent component analysis (ICA) under the same conditions. The accuracy of source estimation is evaluated through a criterion, called again the signal-to-noise-ratio (SNR). The first experiment shows that our proposed method gives accurate estimation of sources in the standard case of independent components, with performance around 27 dB in term of SNR. In the second experiment, we show the capability of the proposed algorithm to successfully separate two noisy mixtures of dependent source components - with classical criterion devoted to the independent case - fails, and that our method is able to deal with the dependent case with good performance.

Conclusions

In this work, we focus specifically on the separation of the ECG signal sources taken from skin two electrodes located on a pregnant woman’s body. The ECG separation is interpreted as a noisy linear BSS problem with instantaneous mixtures. Firstly, a denoising step is required to reduce the noise due to motion artifacts using a BTV filter as a very effective one-pass filter for denoising. Then, we use the Kullbak-Leibler divergence between copula densities to separate the fetal heart rate from the mother one, for both independent and dependent cases.

     

Streptomonospora algeriensis sp. nov., a halophilic actinomycete isolated from soil in Algeria

A halophilic actinomycete strain, designated H27^T, was isolated from a soil sample collected from a hypersaline habitat in Djelfa Province (North-Central Algeria), and then investigated using a polyphasic taxonomic approach. The strain was observed to produce poor aerial mycelium, which formed short chains of oval to cylindrical-shaped spores at maturity, and non fragmented substrate mycelium. The optimum NaCl concentration for growth was found to be 10–15 % (w/v) and the optimum growth temperature and pH were found to be 28–37 °C and 6–7, respectively. The diagnostic diamino acid in the cell-wall peptidoglycan was identified as meso-diaminopimelic acid. The predominant menaquinones of strain H27^T were identified as MK-11 (H₄) and MK-10 (H₆). The major fatty acids were found to be iso-C_16:0, anteiso-C_17:0, 10 methyl C_17:0 and 10 methyl C_16:0. The diagnostic phospholipids detected were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. The chemotaxonomic properties of strain H27^T are consistent with those shared by members of the genus Streptomonospora. 16S rRNA gene sequence analysis indicated that strain H27^T is most closely related to Streptomonospora alba DSM 44588^T (98.8 %) and Streptomonospora flavalba DSM 45155^T (98.7 %) whereas the DNA–DNA relatedness values between strain H27^T and the two type strains were 17.1 and 57.9 %, respectively. Based on the combined genotypic and phenotypic evidence, it is proposed that strain H27^T should be classified as representative of a novel species, for which the name Streptomonospora algeriensis sp. nov. is proposed. The type strain is H27^T (=DSM 45604^T =CCUG 63369^T =MTCC 11563^T).     

Mzabimyces algeriensis gen. nov., sp. nov., a halophilic filamentous actinobacterium isolated from a Saharan soil,and proposal of Mzabimycetaceae fam. nov.

Three halophilic mycelium-forming actinobacteria, strains H195^T, H150 and H151, were isolated from a Saharan soil sample collected from Béni-isguen in the Mzab region (Ghardaïa, South of Algeria) and subjected to a polyphasic taxonomic characterisation. These strains were observed to show an aerial mycelium differentiated into coccoid spore chains and fragmented substrate mycelium. Comparative analysis of the 16S rRNA gene sequences revealed that the highest sequence similarities were to Saccharopolyspora qijiaojingensis YIM 91168^T (92.02 % to H195^T). Phylogenetic analyses showed that the strains H195^T, H150 and H151 represent a distinct phylogenetic lineage. The cell-wall hydrolysate was found to contain meso-diaminopimelic acid, and the diagnostic whole-cell sugars were identified as arabinose and galactose. The major cellular fatty acids were identified as iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic phospholipid detected was phosphatidylcholine and MK-9 (H₄) was found to be the predominant menaquinone. The genomic DNA G+C content of strain H195^T was 68.2 mol%. On the basis of its phenotypic features and phylogenetic position, we propose that strain H195^T represents a novel genus and species, Mzabimyces algeriensis gen. nov., sp. nov., within a new family, Mzabimycetaceae fam. nov. The type strain of M. algeriensis is strain H195^T (=DSM 46680^T = MTCC 12101^T).