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.
- MeSH
- RNA, Bacterial chemistry genetics isolation & purification metabolism MeSH
- Nitrogen-Fixing Bacteria genetics metabolism MeSH
- Centrifugation, Density Gradient MeSH
- DNA, Bacterial chemistry genetics isolation & purification metabolism MeSH
- Nitrogen Fixation genetics physiology MeSH
- Isotope Labeling methods MeSH
- Nitrogen Isotopes metabolism MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Keywords
- FOLANDROL,
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Fertilization in Vitro methods MeSH
- Sperm Injections, Intracytoplasmic methods MeSH
- Humans MeSH
- Magnetics MeSH
- Infertility, Male complications MeSH
- Oligospermia drug therapy MeSH
- Dietary Supplements MeSH
- Cell Separation methods MeSH
- Spermatozoa cytology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
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
- Centrifugation, Density Gradient methods MeSH
- Cryopreservation methods MeSH
- Sperm Motility physiology MeSH
- Silicon Dioxide chemistry MeSH
- Povidone chemistry MeSH
- Proteomics MeSH
- Fishes physiology MeSH
- Spermatozoa physiology MeSH
- Semen Preservation methods MeSH
- Cell Survival physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Blastocystis growth & development pathogenicity MeSH
- Blastocystis Infections diagnosis parasitology MeSH
- Centrifugation, Density Gradient MeSH
- Feces parasitology MeSH
- Gerbillinae MeSH
- Rats MeSH
- Humans MeSH
- Disease Models, Animal * MeSH
- Murinae MeSH
- Mice MeSH
- Disease Susceptibility MeSH
- Specific Pathogen-Free Organisms MeSH
- Rats, Wistar MeSH
- Health Status MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Comparative Study 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.
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.
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.
- MeSH
- Cell Membrane drug effects metabolism MeSH
- Killer Cells, Natural cytology metabolism MeSH
- Centrifugation, Density Gradient MeSH
- Chemotaxis, Leukocyte physiology MeSH
- Detergents pharmacology MeSH
- Cells, Cultured MeSH
- NK Cell Lectin-Like Receptor Subfamily C metabolism MeSH
- NK Cell Lectin-Like Receptor Subfamily K physiology MeSH
- Humans MeSH
- RNA, Small Interfering pharmacology MeSH
- Membrane Microdomains drug effects physiology MeSH
- Actin Cytoskeleton physiology MeSH
- Microfilament Proteins antagonists & inhibitors genetics physiology MeSH
- Multiprotein Complexes MeSH
- Proteome MeSH
- Receptors, Immunologic metabolism MeSH
- RNA Interference MeSH
- Signal Transduction immunology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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).
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
- Adenosine Triphosphate pharmacology MeSH
- Centrifugation, Density Gradient MeSH
- Cell Fractionation methods MeSH
- Glucosylceramidase metabolism MeSH
- HEK293 Cells MeSH
- HeLa Cells MeSH
- Intracellular Membranes drug effects metabolism MeSH
- Acids metabolism MeSH
- Humans MeSH
- Lysosomes drug effects metabolism MeSH
- Subcellular Fractions drug effects metabolism MeSH
- Blotting, Western MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Enzyme Activation MeSH
- Lymphocyte Activation MeSH
- Leukocyte Common Antigens metabolism MeSH
- Cell Membrane metabolism MeSH
- CD4-Positive T-Lymphocytes immunology MeSH
- Centrifugation, Density Gradient MeSH
- Detergents pharmacology MeSH
- Membrane Microdomains metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Signal Transduction MeSH
- Protein Transport MeSH
- Lymphocyte Specific Protein Tyrosine Kinase p56(lck) immunology metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
