Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Neutral glycosphingolipids and gangliosides were isolated from3.7 x 10⁹ primary bovine aortic endothelial cells and structurallycharacterized by immunological and chemical methods. Glucosyl-and lactosylceramide were detected as the main neutral glycosphingolipids(28% and 40% of total orcinol stain, respectively); LcOse₃Cerand nLcOse₄Cer were expressed to somewhat minor amounts (16%and 10% of total orcinol stain, respectively), and nLcOse₆Ceroccurred only in trace quantities. No neutral glycosphingolipidsof the ganglio-series (GgOse₃Cer and GgOse₄Cer) and the globo-series(GbOse₄Cer and the Forssman antigen) have been detected; onlytraces of GbOse₃Cer were identified by TLC immunostaining. PositiveCD15 bands obtained by TLC overlay with anti-Galβ1–4(Fucl-3)GlcNAcβ1-Rantibody indicated the presence of lipid bound Lewis^x antigen,whereas the isomeric Lewis^a structure (Galβ1–3(Fuc1–4)GlcNAcβ1-R)was not detectable. G_M3 substituted with Neu5Gc and Neu5Ac ina 2:1 ratio was the major ganglioside comprising about 95% withinthe whole ganglioside fraction. G_M3-structures were furthercharacterized by FAB-MS and GC-MS of the native compounds andtheir permethylated derivatives. C₁₈-sphingosine was the onlylong chain base, whereas variation occurred due to C_24:0,24:1and C₁₆ fatty acids. Terminally 2–3 sialylated neolacto-seriesgangliosides with nL-cOse₄- and nLcOse₆Cer (<5% of totalresordnol stain) were found in almost equal quantities, whereasno 2–6 sialylated counterparts were detected. Fucosylatedgangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis^x,sialyl Lewis^a, and VIM-2 antigen) and sulfoglucuronyl-neolactoseries structures with HNK-1 epitope were not detectable inthe acidic glycosphingoiipid fraction by TLC immunostaining.Gangliotetraose-type gangliosides G_M1 and G_D1a (<1 % of totalresorcinol stain) as well as traces of G_D1b and G_T1b have beendistinctly identified by combined choleragenoid-TLC-immunostainingand previous neur-aminidase treatment.The expression of dominantglycosphingolipids lactosylceramide and G_M3(Neu5Gc) was provedby indirect immunofiuorescence microscopy of cell layers grownin chamber slides, each showing different plasma membrane andsubcellular distribution patterns. The results provide the basisfor investigation of the role of glycosphingolipids as cellsurface antigens of cellular interaction as well as receptorsfor blood components and mac-romolecules of the extracellularmatrix. gangliosides neutral glycosphingolipids antibodies Lewis^x antigen TLC immunostaining     

"Candidatus Bacilloplasma," a novel lineage of Mollicutes associated with the hindgut wall of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda)

Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio scaber (Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class Mollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes. The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of P. scaber and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name "Candidatus Bacilloplasma" has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. scaber.     

Contact allergy to special and standard allergens in patients with venous ulcers

The aim of the study was to determine the prevalence of contact sensitivity in patients with leg ulcers, and possible difference in the rate of contact hypersensitivity to standard series of allergens used in patch testing, and to particular topical agents used in local therapy of leg ulcers in special series, patients with and without atopy. The study included 60 patients, 45 female and 15 male, aged 37-85 (mean 68.37 female and 51.13 male), 30 of them with and 30 without allergic contact dermatitis (ACD) of the leg (control group). The mean duration of leg ulceration was 5.62 years. The two groups of patients underwent testing to standard series allergens and target series allergens including mupirocin, bepanthene, silver sulfadiazine, chloramphenicol + clostridiopeptidase, betamethasone dipropionate, hydrocortisone + oxytetracycline, momethasone, alginate, hydrocolloid, lanolin, pyrogallol, Vaseline, permanganate, Rivanol, povidone-iodine, gentamicin, i.e. local agents most frequently used by the patients. Contact allergic hypersensitivity to standard series allergens was demonstrated in 25 patients with a total of 49 positive reactions and a mean of 1.6 reactions per patient. Positive reactions were most commonly recorded to balsam of Peru, fragrance mix and neomycin sulfate. There were 12 positive reactions to target series allergens, mean 0.4 reactions per patient. Forty-five positive reactions, mean 0.1 reactions per patient, were recorded in the control group. Positive reactions were most commonly demonstrated to corticosteroid ointments, lanolin and bepanthene. Study results did not confirm a statistically significantly higher rate of sensitization to particular topical agents frequently used in the treatment of patients with venous ulcers. Patch testing to standard and special series allergens should be performed in case of prolonged leg ulcer epithelization.     

Dwell-time distribution analysis of polyprotein unfolding using force-clamp spectroscopy

Using the recently developed single molecule force-clamp technique we quantitatively measure the kinetics of conformational changes of polyprotein molecules at a constant force. In response to an applied force of 110 pN, we measure the dwell times of 1647 unfolding events of individual ubiquitin modules within each protein chain. We then establish a rigorous method for analyzing force-clamp data using order statistics. This allows us to test the success of a history-independent, two-state model in describing the kinetics of the unfolding process. We find that the average unfolding trajectory is independent of the number of protein modules N in each trajectory, which varies between 3 and 12 (the engineered protein length), suggesting that the unfolding events in each chain are uncorrelated. We then derive a binomial distribution of dwell times to describe the stochastic dynamics of protein unfolding. This distribution successfully describes 81% of the data with a single rate constant of alpha = 0.6 s(-1) for all N. The remainder of the data that cannot be accounted for suggests alternative unfolding barriers in the energy landscape of the protein. This method investigates the statistical features of unfolding beyond the average measurement of a single rate constant, thus providing an attractive alternative for measuring kinetics by force-clamp spectroscopy.     

Contour length and refolding rate of a small protein controlled by engineered disulfide bonds

The introduction of disulfide bonds into proteins creates additional mechanical barriers and limits the unfolded contour length (i.e., the maximal extension) measured by single-molecule force spectroscopy. Here, we engineer single disulfide bonds into four different locations of the human cardiac titin module (I27) to control the contour length while keeping the distance to the transition state unchanged. This enables the study of several biologically important parameters. First, we are able to precisely determine the end-to-end length of the transition state before unfolding (53 Angstrom), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 Angstrom). Second, the measured contour length per amino acid from five different methods (4.0 +/- 0.2 Angstrom) is longer than the end-to-end length obtained from the crystal structure (3.6 Angstrom). Our measurement of the contour length takes into account all the internal degrees of freedom of the polypeptide chain, whereas crystallography measures the end-to-end length within the "frozen" protein structure. Furthermore, the control of contour length and therefore the number of amino acids unraveled before reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (tau(F)). We find that both a power law scaling tau(F) lambda n(lambda) with lambda = 4.4, and an exponential scaling with n(0.6) fit the data range, in support of different protein-folding scenarios.     

Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin

Single-protein force experiments have relied on a molecular fingerprint based on tethering multiple single-protein domains in a polyprotein chain. However, correlations between these domains remain an issue in interpreting force spectroscopy data, particularly during protein folding. Here we first show that force-clamp spectroscopy is a sensitive technique that provides a molecular fingerprint based on the unfolding step size of four single-monomer proteins. We then measure the force-dependent unfolding rate kinetics of ubiquitin and I27 monomers and find a good agreement with the data obtained for the respective polyproteins over a wide range of forces, in support of the Markovian hypothesis. Moreover, with a large statistical ensemble at a single force, we show that ubiquitin monomers also exhibit a broad distribution of unfolding times as a signature of disorder in the folded protein landscape. Furthermore, we readily capture the folding trajectories of monomers that exhibit the same stages in folding observed for polyproteins, thus eliminating the possibility of entropic masking by other unfolded modules in the chain or domain-domain interactions. On average, the time to reach the I27 folded length increases with increasing quenching force at a rate similar to that of the polyproteins. Force-clamp spectroscopy at the single-monomer level reproduces the kinetics of unfolding and refolding measured using polyproteins, which proves that there is no mechanical effect of tethering proteins to one another in the case of ubiquitin and I27.     

Isoflurane preconditioning uncouples mitochondria and protects against hypoxia-reoxygenation

Ischemic cardiac injury can be substantially alleviated by exposing the heart to pharmacological agents such as volatile anesthetics before occurrence of ischemia-reperfusion. A hallmark of this preconditioning phenomenon is its memory, when cardioprotective effects persist even after removal of preconditioning stimulus. Since numerous studies pinpoint mitochondria as crucial players in protective pathways of preconditioning, the aim of this study was to investigate the effects of preconditioning agent isoflurane on the mitochondrial bioenergetic phenotype. Endogenous flavoprotein fluorescence, an indicator of mitochondrial redox state, was elevated to 195 ± 16% of baseline upon isoflurane application in intact cardiomyocytes, indicating more oxidized state of mitochondria. Isoflurane treatment also elicited partial dissipation of mitochondrial transmembrane potential, which remained depolarized even after anesthetic withdrawal (tetramethylrhodamine fluorescence intensity declined to 83 ± 3 and 81 ± 7% of baseline during isoflurane exposure and washout, respectively). Mild uncoupling, with preserved ATP synthesis, was also detected in mitochondria that were isolated from animals that had been previously preconditioned by isoflurane in vivo, revealing its memory nature. These mitochondria, after exposure to hypoxia and reoxygenation, exhibited better preserved respiration and ATP synthesis compared with mitochondria from nonpreconditioned animals. Partial mitochondrial depolarization was paralleled by a diminished Ca²⁺ uptake into isoflurane-treated mitochondria, as indicated by the reduced increment in rhod-2 fluorescence when mitochondria were challenged with increased Ca²⁺ (180 ± 24 vs. 258 ± 14% for the control). In conclusion, isoflurane preconditioning elicits partial mitochondrial uncoupling and reduces mitochondrial Ca²⁺ uptake. These effects are likely to reduce the extent of the mitochondrial damage after the hypoxic stress. cardioprotection; uncoupling     

Identification of Monosialylated N-glycoforms in the CDG Urinome by Ion Mobility Tandem Mass Spectrometry: The Potential for Clinical Applications

Introduction

A novel approach of ion mobility tandem mass spectrometry (IMS-MS/MS) is applied to analysis of human glycourinome to obtain carbohydrate pattern data of congenital disorders of glycosylation patient. Overlapping of the complex carbohydrate mass range landscape has been highly reduced upon IMS-MS procedure, allowing more efficient identification by mapping and sequencing of glycan precursor ions, following their separation by mobility, according to difference in drift time through the traveling wave IMS cell. Intact and truncated N- and O-glycan structures modified by sialylation and fucosylation were identified according to their drift time separated molecular ions and submitted to fragmentation in a narrow mass window.

IMS CID MS/MS Analysis

The fragmentation spectra generated from the IMS separated precursor ions contain series of fragment ions maintaining the same mobility as their parent ions, and the assignment accuracy can be significantly enhanced.

Conclusion

According to the specific fragment ion patterns, carbohydrate epitopes described to be involved in pathological processes were assigned. A high potential of this glycomics-based strategy for clinical applications can be presented.     

Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.