Nitrogen fixation and assimilation processes are vital to the functioning of any ecosystem. Nevertheless, studying these processes using 15N-based stable isotope probing was so far limited because of technical challenges related to the relative rarity of nitrogen in nucleic acids and proteins compared to carbon, and because of its absence in lipids. However, the recent adoption of high-throughput sequencing and statistical modelling methods to SIP studies increased the sensitivity of the method and enabled overcoming some of the challenges. This chapter describes in detail how to perform DNA- and RNA-SIP using 15N.

• Klíčová slova
• 15N, Amplicon sequencing, BNF, DNA-SIP, Diazotrophs, Nitrogen, RNA-SIP,
• MeSH
• bakteriální RNA chemie   genetika   izolace a purifikace   metabolismus   MeSH
• bakterie fixující dusík genetika   metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• DNA bakterií chemie   genetika   izolace a purifikace   metabolismus   MeSH
• fixace dusíku genetika   fyziologie   MeSH
• izotopové značení metody   MeSH
• izotopy dusíku metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální RNA MeSH
• DNA bakterií MeSH
• izotopy dusíku MeSH
• Nitrogen-15 MeSH Prohlížeč

In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.

• MeSH
• centrifugace - gradient hustoty metody   MeSH
• kryoprezervace metody   MeSH
• motilita spermií fyziologie   MeSH
• oxid křemičitý chemie   MeSH
• povidon chemie   MeSH
• proteomika MeSH
• ryby fyziologie   MeSH
• spermie fyziologie   MeSH
• uchování spermatu metody   MeSH
• viabilita buněk fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

Blastocystis is a common inhabitant of the human gut, colonizing at least one billion people at a prevalence ranging from <10% to 100% in healthy human populations globally. The majority of carriers remain asymptomatic, suggesting that Blastocystis is largely a commensal, though Blastocystis has also been implicated in disease in some people. However, there are no in vivo model systems in which to experimentally test the impact of Blastocystis on mammalian hosts and the gut ecosystem and determine which factors underlie these variable clinical outcomes. We evaluated a rat model for sustaining of a human-derived Blastocystis ST1 and assess colonization success and longevity. Because of the broad host range of Blastocystis, we compared the rat with three other rodent species to establish the reproducibility of our method. Blastocystis was introduced by esophageal gavage and colonization success evaluated by Blastocystis culture. Culture was also used to determine that all animals were negative prior to colonization and negative controls remain Blastocystis-free. In this study, Blastocystis ST1 established in 100% of the outbred rats (Rattus norvegicus) and gerbils (Meriones unguiculatus) challenged. Rats were colonized asymptomatically for more than one year, but Blastocystis ST1 was not transmitted between rats. Mus musculus strain CD1 and Mastomys coucha were not susceptible to Blastocystis ST1. Thus, rats appear to be a suitable in vivo model for studies of Blastocystis ST1, as do gerbils though testing was less extensive. This work lays the foundation for experimental work on the role of Blastocystis in health and disease.

• Klíčová slova
• Blastocystis ST1, Colonization longevity, Experimental colonization, In vivo model, Rat, Susceptibility to colonization,
• MeSH
• Blastocystis růst a vývoj   patogenita   MeSH
• blastocystóza diagnóza   parazitologie   MeSH
• centrifugace - gradient hustoty MeSH
• feces parazitologie   MeSH
• Gerbillinae MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• Murinae MeSH
• myši MeSH
• náchylnost k nemoci MeSH
• organismy bez specifických patogenů MeSH
• potkani Wistar MeSH
• zdravotní stav MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• srovnávací studie MeSH

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

• MeSH
• analýza spermatu MeSH
• centrifugace - gradient hustoty veterinární   MeSH
• kryoprezervace veterinární   MeSH
• separace buněk metody   veterinární   MeSH
• uchování spermatu veterinární   MeSH
• vysoká zvěř fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.

• MeSH
• Arabidopsis cytologie   MeSH
• časové faktory MeSH
• centrifugace - gradient hustoty ekonomika   metody   MeSH
• oxid křemičitý chemie   MeSH
• povidon chemie   MeSH
• pyl cytologie   MeSH
• separace buněk ekonomika   metody   MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis.

• Klíčová slova
• chemotaxis, membrane rafts,
• MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buňky NK cytologie   metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• chemotaxe leukocytů fyziologie   MeSH
• detergenty farmakologie   MeSH
• kultivované buňky MeSH
• lektinové receptory NK-buněk - podrodina C metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina K fyziologie   MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• membránové mikrodomény účinky léků   fyziologie   MeSH
• mikrofilamenta fyziologie   MeSH
• mikrofilamentové proteiny antagonisté a inhibitory   genetika   fyziologie   MeSH
• multiproteinové komplexy MeSH
• proteom MeSH
• receptory imunologické metabolismus   MeSH
• RNA interference MeSH
• signální transdukce imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• detergenty MeSH
• HCST protein, human MeSH Prohlížeč
• KLRK1 protein, human MeSH Prohlížeč
• LCP1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina C MeSH
• lektinové receptory NK-buněk - podrodina K MeSH
• malá interferující RNA MeSH
• mikrofilamentové proteiny MeSH
• multiproteinové komplexy MeSH
• proteom MeSH
• receptory imunologické MeSH

We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• centrifugace - gradient hustoty MeSH
• frakcionace buněk metody   MeSH
• glukosylceramidasa metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• intracelulární membrány účinky léků   metabolismus   MeSH
• kyseliny metabolismus   MeSH
• lidé MeSH
• lyzozomy účinky léků   metabolismus   MeSH
• subcelulární frakce účinky léků   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• glukosylceramidasa MeSH
• kyseliny MeSH

More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

• Klíčová slova
• Grow yeast cultures, Polysome profile analysis, Sucrose density gradient centrifugation, Western and Northern blotting, Yeast whole cell extracts (WCEs),
• MeSH
• centrifugace - gradient hustoty metody   MeSH
• northern blotting metody   MeSH
• polyribozomy chemie   genetika   MeSH
• Saccharomyces cerevisiae chemie   cytologie   genetika   MeSH
• sacharosa chemie   MeSH
• western blotting metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• sacharosa MeSH

Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

• MeSH
• buněčná membrána metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• cytosol metabolismus   MeSH
• HEK293 buňky MeSH
• hormon uvolňující thyreotropin metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• molekulová hmotnost MeSH
• myši MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 izolace a purifikace   metabolismus   MeSH
• receptory thyroliberinu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hormon uvolňující thyreotropin MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory thyroliberinu MeSH

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

• MeSH
• aktivace enzymů MeSH
• aktivace lymfocytů MeSH
• antigeny CD45 metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• centrifugace - gradient hustoty MeSH
• detergenty farmakologie   MeSH
• membránové mikrodomény metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• signální transdukce * MeSH
• transport proteinů MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD45 MeSH
• detergenty MeSH
• PTPRC protein, human MeSH Prohlížeč
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH