Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

Stone surface topography of Prague historic monuments over the centuries

Stonemasons tool traces on stone surface are undoubtably a significant part of monument historic value. As any other profession, stonemasons craft develops over time and has its own specifics in every period. Each work has its own unique pattern and bears traces of individual stonemason workshops, tradition, etc. The study of building stone cutting is based on evolving methods of mechanoscopy and analytical traceology. Stone traceology deals with traces in material and the subsequent reconstruction of tools and the processes of stoneworking. The interpretation of data in terms of determining the actual trace is called a mechanoscopy. When studying a stone surface, the latest 3D modelling technology is used with subsequent analyses by means of Global Mapper software. The use of these methods enables reconstruction of the craftsman’s tools. The presented article is an extract of a study that systematically maps the stone cutting work in the territory of Prague from the oldest buildings to the present day. For example, stonework in 9th century Prague was gradually evolving from simple stone block modelling to sophisticated cutting of blocks in the 12th century. This information can be very useful in the process of monument care

Distribution of Sulfate-Reducing Bacteria in the Environment: Cryopreservation Techniques and Their Potential Storage Application

Sulfate-reducing bacteria (SRB) are a heterogeneous group of anaerobic microorganisms that play an important role in producing hydrogen sulfide not only in the natural environment, but also in the gastrointestinal tract and oral cavity of animals and humans. The present review was written with the inclusion of 110 references including the time period from 1951 to 2021. The following databases were evaluated: Web of Science, Scopus and Google Scholar. The articles chosen to be included in the review were written mainly in the English and Czech languages. The molecular mechanisms of microbial cryoprotection differ depending on the environment where microorganisms were initially isolated. It was observed that the viability of microorganisms after cryopreservation is dependent on a number of factors, primarily colony age, amount of inoculum, cell size or rate of cooling, and their molecular inventory. Therefore, this paper is devoted to assessing the performance and suitability of various cryopreservation methods of intestinal bacteria, including molecular mechanisms of their protection. In order to successfully complete the cryopreservation process, selecting the correct laboratory equipment and cryopreservation methodology is important. Our analysis revealed that SRB should be stored in glass vials to help mitigate the corrosive nature of hydrogen sulfide, which can affect their physiology on a molecular level. Furthermore, it is recommended that their storage be performed in distilled water or in a suspension with a low salt concentration. From a molecular biological and bioengineering perspective, this contribution emphasizes the need to consider the potential impact associated with SRB in the medical, construction, and environmental sectors

Detection and Characterization of Phosphorylation, Glycosylation, and Fatty Acid Bound to Fetuin A in Human Blood

The hepatokine fetuin A (Fet A) has been associated with diverse pathological states such as insulin resistance, type 2 diabetes, macrovascular disease, and systemic ectopic and vascular calcification. Fet A may also play a role in tumor growth and metastasis. The biological activity of Fet A may be affected by various modifications, including phosphorylation, O- and N-glycosylation and fatty acid binding. We developed an antibody-based assay for the detection of Fet A phosphorylated at serine 312. Fatty acid pattern was determined by gas chromatography. Using the antibody, we found that the phosphorylation was stable in human plasma or serum at room temperature for 8 h. We observed that Fet A is present in several glycosylation forms in human plasma, but the extent of Ser312 phosphorylation was not associated with glycosylation. The phosphorylation pattern did not change during an oral glucose tolerance test (0-120 min). We further found that human Fet A binds preferentially saturated fatty acids (>90%) at the expense of mono- and poly-unsaturated fatty acids. Our results indicate that different molecular species of Fet A are present in human plasma and that these different modifications may determine the different biological effects of Fet A

Detection and characterization of phosphorylation, glycosylation, and fatty acid bound to fetuin A in human blood.

The hepatokine fetuin A (Fet A) has been associated with diverse pathological states such as insulin resistance, type 2 diabetes, macrovascular disease, and systemic ectopic and vascular calcification. Fet A may also play a role in tumor growth and metastasis. The biological activity of Fet A may be affected by various modifications, including phosphorylation, O- and N-glycosylation and fatty acid binding. We developed an antibody-based assay for the detection of Fet A phosphorylated at serine 312. Fatty acid pattern was determined by gas chromatography. Using the antibody, we found that the phosphorylation was stable in human plasma or serum at room temperature for 8 h. We observed that Fet A is present in several glycosylation forms in human plasma, but the extent of Ser312 phosphorylation was not associated with glycosylation. The phosphorylation pattern did not change during an oral glucose tolerance test (0-120 min). We further found that human Fet A binds preferentially saturated fatty acids (>90%) at the expense of mono- and poly-unsaturated fatty acids. Our results indicate that different molecular species of Fet A are present in human plasma and that these different modifications may determine the different biological effects of Fet A