Discontinuous Density Gradient Centrifugation Research Articles

16FERTILIZING POTENTIAL OF STORED TURKEY SPERM

Considerable interest exists in prolonging the viability and duration of the fertilizing capacity of turkey sperm. Commercially available extenders for turkey semen are unable to maintain sperm survival for longer than a few hours and attempts to develop a suitable cryopreservation medium have met with limited success. The objective of the present study was to investigate the use of andrology products designed and optimized for turkey sperm (NidaCon International). In an insemination trial on a commercial turkey farm, semen was collected by abdominal massage and was immediately extended in 4mL of either Turkey Semen Extend (NidaCon International, Gothenburg, Sweden) or Beltsville (Continental Plastic Corp, Delavan, WI). The extended semen (approximately 20μL, representing a dose of approximately 70 million sperm) was used immediately for artificial insemination, with each bird being inseminated once weekly. The two groups of birds were housed in different buildings, with different semen donors for each. Eggs were candled after 5 days. Preliminary results showed that fertilization and hatching rates were similar in the two groups: Group 1 (Turkey Semen Extend) 966 eggs, 84.4% fertilized, 78.7% hatched; Group 2 (Beltsville) 966 eggs, 86.6% fertilized, 79% hatched. A second trial over a longer period showed similar results: Group 3 (Turkey Semen Extend) 18,450 eggs, 93.3% fertilized, 84.6% hatched; Group 4 (Beltsville) 40,873 eggs, 92% fertilized, 84.2% hatched. In a second experiment, turkey semen, extended as described above, was transported to the laboratory in the dark at ca. 34°C. Sperm motility was assessed subjectively at several time points. After 2 hours’ storage, at least 50% of the sperm in Turkey Semen Extend were still motile, compared to only approximately 5% of the sperm in Beltsville medium. Aliquots of semen in Turkey Semen Extend were processed by discontinuous density gradient centrifugation on Turkey Gradient (NidaCon International). At least 40% motility was observed after 24 hours’ storage at room temperature. In conclusion, the new Turkey Semen Extender and Turkey Density Gradient offer exciting possibilities for improving the viability of stored turkey sperm for insemination. Future studies will assess the fertility of both unprocessed stored turkey sperm in Turkey Semen Extend, and gradient-prepared stored turkey sperm.

Isolation and partial characterisation of the plasma and outer acrosomal membranes of goat spermatozoa

In order to achieve successful fertilization, the essential steps include maturation, species-specific binding and fusion of gametes followed by activation of the ovum. Implicit in this are a variety of molecular events, the most important among them being the receptor–ligand interaction. A protocol is described to isolate highly enriched fractions of goat sperm plasma and the outer acrosomal membranes and present data on the ultrastructure and enzyme assays of these membrane domains. The sperm, washed with 1.3 M sucrose sedimentation, was sonicated and subjected to discontinuous sucrose density gradient centrifugation, which allowed the separation of isolated plasma and the outer acrosomal membranes in a single step. While the isolated plasma membranes formed vesicles, the cap shaped acrosomal segments retained its characteristic morphology, as was seen in the ultrastructure. The plasma membrane fraction was relatively pure and free of any contamination of other cell organelles. The matrix associated with the isolated acrosomal membranes gave it a relatively higher density and a crescent shape. Enzyme assays showed high alkaline phosphatase activity in the plasma membrane fraction, with low activity of the acrosome bound enzymes. The acrosomal fraction had a higher specific activity of acrosin and hyaluronidase and a low alkaline phosphatase activity. Suggesting that both the membranes were pure. Membranes, thus purified could be used as initial material for elucidating the lipid as well as the antigenic composition and in understanding the mechanism involved in capacitation, gametic interaction and acrosome reaction, which eventually lead to fertilization.

Endotoxin-Free Purification of Monocytes for Dendritic Cell Generation via Discontinuous Density Gradient Centrifugation Based on Diluted Ficoll-Paque Plus®

Background: For the investigation of the biology of human monocyte-derived dendritic cells (DCs), and also for further clinical use of these cells, it is important to separate large numbers of monocytes without isolation-induced activation. Methods: Monocytes were purified from mononuclear cells (MNCs) by density gradient centrifugation using Ficoll-Paque Plus^® adjusted to a density of 1,068 g/ml by dilution with phosphate-buffered saline (PBS) and compared to Percoll^® isolated monocytes. DCs were generated by culturing monocytes with interleukin (IL)-4 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), and they were functionally tested for IL-12 p70 production by stimulation with lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). Results: Density gradient centrifugation using diluted Ficoll-Paque Plus^® resulted in a monocyte purity of more than 75% and a recovery of 70%. DCs derived from such monocytes produced high amounts of IL-12 p70 in response to LPS plus IFN-γ, thus demonstrating an important functional capacity. Conclusion: Since Ficoll-Paque Plus^® is commercially available in an endotoxin-free form, this modified Ficoll gradient offers a simple modality for isolating large amounts of monocytes under endotoxin-free conditions without introducing further potential monocyte-activating agents or procedures.

Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.

Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.

Reactive oxygen species (ROS)-induced damage of membrane phospholipids and DNA in human spermatozoa has been implicated in the pathogenesis of male infertility. In this study, variations in ROS production, DNA structure (as measured by the sperm chromatin structure assay) and lipid composition, were studied in human spermatozoa at different stages of maturation. Sperm subsets were isolated by discontinuous density gradient centrifugation of semen samples obtained from healthy donors and from infertility patients. DNA damage and ROS production were highest in immature spermatozoa with cytoplasmic retention and abnormal head morphology, and lowest in mature spermatozoa. Docosahexaenoic acid and sterol content were highest in immature germ cells and immature spermatozoa, and lowest in mature spermatozoa. The relative proportion of ROS-producing immature spermatozoa in the sample was directly correlated with DNA damage in mature spermatozoa, and inversely correlated with the recovery of motile spermatozoa. There was no correlation between DNA damage and sperm morphology in mature spermatozoa. The high levels of ROS production and DNA damage observed in immature spermatozoa may be indicative of derangements in the regulation of spermiogenesis. DNA damage in mature spermatozoa may be the result of oxidative damage by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis.

Interference study should be performed for every protein measurement method used

It is particularly important, when analyzing biological material, for the measurement procedure to be specific to the analyte and not to suffer interference by the matrix effect. Tissue fraction studies also require rapid and accurate methods to estimate the concentration of protein in solutions as well as many measurement methods used in medical laboratories. The design of this study is based on a comparison of the Lowry and the bicinchoninic acid (BCA) methods for the measurement of the total protein concentrations of rat liver subcellular fractions. In our experiment, subcellular fractions enriched in peroxisomes (POs) obtained by differential centrifugation were then further separated by means of density gradient centrifugation. We performed the protein measurement assays on all fractions obtained during the purification steps. The protein contents of the fractions obtained were determined by the two methods. The method comparison statistics were performed by linear Deming regression analysis and Altman and Bland bias plot. The regression equation was unacceptable, indicating that the last three fractions separated by means of Nycodenz discontinuous density gradient centrifugation gave remarkably divergent results. For the Lowry method, the Nycodenz effect could not be eliminated with the use of interference blank. In addition to Nycodenz, the potentially interfering compound used in the isolation procedure as isolation medium was 3-(morpholino)propane sulfonic acid (MOPS). In decreased concentrations of MOPS (10 mM), interference blank should be used for correct measurement with Lowry, but in practical use 10 mM does not provide buffering potency. In the BCA method, interference blank correction seemed to eliminate the measurement error in all concentrations of Nycodenz. There was no MOPS effect on the BCA measurement assay. Referring to deviations as sample-inherent matrix effects, we concluded that not only one, but more measurement methods should be used in order to make a correct protein measurement.

Separation of distinct compartments of rice Golgi complex by sucrose density gradient centrifugation

We studied the separation of distinct compartments of the Golgi complex in rice and tobacco employing the discontinuous sucrose density gradient centrifugation technique with EDTA or MgCl 2. In the presence of EDTA, rice Golgi marker enzymes, nucleoside diphosphatase and N-acetylglucosaminyltransferase were fractionated in the 28 and 33% sucrose fractions, respectively. Peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 that are shown to be located at Golgi complex were broadly distributed around a 31.5% sucrose density. Under a MgCl 2-supplemented condition, the peak of nucleoside diphosphatase was shifted to 29 and 37% sucrose and N-acetylglucosaminyltransferase was distributed in the 37% sucrose fraction. The distribution of peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 were also shifted to the 34.5% sucrose fraction. Furthermore, glucan synthase-I and UDP-glucose pyrophosphorylase were fractionated in the 26.5% sucrose fraction, regardless of the presence or absence of MgCl 2. These results indicate that there exist at least four distinct compartments of the rice Golgi complex. The tobacco Golgi marker enzymes and Golgi-localized proteins were distributed in the 30–31% sucrose fractions in the presence of EDTA and slightly shifted to the 32–33% sucrose fractions under a MgCl 2-supplemented condition. These results suggest that the sedimentation behavior of compartments of the rice Golgi complex in the gradients is specific for rice cells.

Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in Ras.

Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction.

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism.

Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity. To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [51Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing. Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate. These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.

Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E 2) and progesterone (P 4) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E 2 could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P 4 in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E 2 was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P 4 ( P<0.05) in the serum-free cell culture media. The stimulation of P 4 production was greater at higher LH concentration (50 ng/ml) ( P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1–100 ng/ml) showed that FGF-2 inhibited the production of P 4 by LH-stimulated thecal cell cultures ( P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC 50<1 ng/ml at LH-20 ng/ml vs. EC 50>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E 2 production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.

Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse a- and b-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529-539, 2000. © 2000 Wiley-Liss, Inc.

Isolation of fetal nucleated red blood cells from maternal blood.

To find a simple, effective method of isolating fetal cells from maternal peripheral blood for prenatal diagnosis, 45 women were studied with their gestation being 6-14 weeks and age 21-30 years. The fetal cells were isolated from maternal blood by using discontinuous density gradient centrifugation. Some of the isolated cells were made smear and counted under the microscope; others were used for predicting fetal sex by PCR amplification of Y chromosome specific DYZ1 gene. The major cells in the upper separation interface were lymphocytes and monocytes, with occasionally seen nucleated red blood cells (NRBC); while those in the middle separation interface were neutrocytes, with NRBC scattering. The ratio of NRBC/nucleated cells was 1.98 +/- 0.28 x 10(-5). There was no significant difference between the first and second trimester (P > 0.05). The amount of isolated fetal cells was sufficient for prenatal genetic diagnosis. Male pregnancy was correctly predicted in 10 out of 13 cases. It is concluded that the method of discontinuous density gradient centrifugation was of considerable importance in the development of non-invasive prenatal genetic diagnosis.

Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.

Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples

The investigation of mitochondrial abnormalities in brain commonly requires isolation of these organelles from small tissue samples. We have modified a mitochondrial isolation procedure based on Percoll density gradient centrifugation to increase the proportion of the total mitochondrial pool recovered while reducing contamination with synaptosomes and related structures containing cytoplasm. Initially, myelin was removed by centrifugation in 12% Percoll in isotonic buffer. The pellet was resuspended, treated with digitonin to break up synaptosomes and similar structures and subjected to discontinuous Percoll density gradient centrifugation. The mitochondrial fraction obtained from this procedure was highly metabolically active and well coupled, exhibiting respiratory control ratios above 5. The recovery of mitochondrial markers using a single rat forebrain as starting material was approximately 18% to 21%. When small tissue samples (approximately 50 mg wet weight) were used as starting material the recovery of the mitochondrial marker was approximately 16%. The ratio of recovery of a mitochondrial marker to the cytoplasmic marker lactate dehydrogenase exceeded 200 in preparations from a single rat forebrain. This is substantially greater than values reported for previously published procedures reflecting both an improved yield of mitochondria and a reduction in cytoplasmic contamination. Themes: Other systems of the CNS Topics: Brain metabolism and blood flow

32] Identification of phosphorylation sites within vertebrate and invertebrate rhodopsin

Publisher Summary Studies of vertebrate and invertebrate rhodopsin have been applicable to our understanding of amplification and desensitization processes of Gprotein-coupled receptors in general. In vertebrate rod outer segments, photoisomerization of rhodopsin (Rho*) initiates activation of hundreds of G-protein (G t ) molecules, which in turn stimulate cGMP phosphodiesterase, resulting in closure of cGMP gated channels in response to the decrease in cytosolic cGMP concentrations. To study the enzymatic and functional roles of Rho phosphorylation, it was necessary to develop protocols for the identification of phosphorylation sites within vertebrate and invertebrate Rho. This chapter describes a method that employs mass spectrometry in conjunction with specific proteolysis, and should be applicable to the study of phosphorylation in other systems. Rho phosphorylation is carried out on rod outer segment (ROS) membranes isolated from fresh bovine retinas by discontinuous sucrose density gradient centrifugation in 10 mM Bis-Tris propane (BTP) buffer, pH 7.5, containing 5 mM MgCl2 and 1 mM ATP at 30° for 0–45 minutes under illumination from a 150-W lamp at a distance of 10 cm. Phosphorylated forms of the peptide could, in principle, be analyzed by tandem mass spectrometry (MS/MS) to identify the specific residues phosphorylated.

IL-2 Enhances Standard IFNγ/LPS Activation of Macrophage Cytotoxicity to Human Ovarian Carcinoma in Vitro: A Potential for Adoptive Cellular Immunotherapy

Objective. The objective was to evaluate the enhancement of human peritoneal macrophage cytotoxic in vitro activity by the addition of interleukin-2 (IL-2) to the standard interferon gama (IFNγ) and lipopolysaccharide (LPS) activation procedure used for cellular adoptive immunotherapy in a human ovarian cancer system. This cytotoxic effect of these activated macrophages was tested on cells from ovarian cancers of various stages, histology type, and grade, both prior to chemotherapy and at recurrence, in ovarian carcinoma cells lines and normal cells. Increased activation of the macrophage may make it a better candidate for intraperitoneal cellular adoptive immunotherapy as a component of ovarian cancer therapy. This was not a study of the mechanism of macrophage killing.Methods. Ascites specimens were collected from 24 ovarian cancer patients at the time of surgery or by paracentesis. The mononuclear cell fraction was isolated by discontinuous density gradient centrifugation and used as a cellular source of peritoneal macrophages (PMs) and primary cultured ovarian cancer cells. PMs were separated by 1-h adhesion followed by intensive washing to remove floating cells. The floating cells were cultured for 24 h which left the cancer cells attached after unattached cells were removed by washing. These cells formed a monolayer of cancer cells, which could be subcultured in 22 patients. The cells from the third to fifth passages were used as target cells without coculture with other cells. PMs were identified by latex ingestion, and their purity after isolation by adhesion culture was tested by flow cytometry and immunofluorescence. PMs were activated by culturing in the presence of IFNγ, with or without IL-2, for 18 h followed by the addition of LPS 6 h prior to use as effector cells in cytotoxicity assays. Ovarian cancer cells of both established cell lines and primary cultures were labeled with 51Cr and utilized as target cells to quantitatively measure PM-mediated cytotoxicity. Ovarian cancer cells were also cocultured with PMs for morphologic observations to provide supporting evidence to the cytotoxicity assays.Results. IL-2 enhances the cytotoxicity of the standard IFNγ/LPS macrophage activation in this system. Peritoneal macrophages so activated are cytotoxic to autologous and allogenic primary cultured ovarian tumors and to ovarian carcinoma cell lines. The macrophages are cytotoxic to cells both prior to treatment and at recurrence, but the data from the few recurrent patients did reach statistical significance. This cytotoxicity is not MHC associated. Normal cells are minimally affected.Conclusions. IL-2 augmented the standard IFNγ/LPS method of activating peritoneal macrophage cell killing of human ovarian cancer cells in this in vitro system. The cell killing occurred with autologous and allogenic tumor cells from patients with primary and possibly recurrent tumors. Activated PMs minimally affected the normal cells tested. This enhanced activation may improve the disappointing results of previous adoptive cellular immunotherapy human trials and should be considered for ovarian cancer clinical trials.

Effects of Exogenous Fatty Acids on H +-ATPase Activity and Lipid Composition of Plasma Membrane Vesicles Isolated from Roots of Barley Seedlings under Salt Stress

Summary The effects of exogenous fatty acids of different saturaturation degrees on the H+-ATPase activity and lipid composition of the plasma membrane (PM) vesicles isolated from the roots of two barley cultivars differing in salt-sensitivity (Jian 4, salt-tolerant; Kepin 7, salt-sensitive) were investigated under 100 mmol L−1 NaCl stress for 2 days. The PM vesicles with high purity were prepared by discontinuous sucrose density gradient centrifugation. The H+-ATPase activities, phospholipid contents and the index of unsaturated fatty acids (IUFA) increased in the PM vesicles isolated from the roots of seedlings of two barley cultivars under NaCl stress. However, there was no obvious change in the IUFA value with exogenous fatty acids of different saturation degrees. Exogenous stearic acid and linoleic acid increased the H+-ATPase activities and phospholipid contents, but their effects on galactolipid contents of the PM of Kepin 7 were different. For Jian 4, treated with linoleic acid, the galactolipid content increased by 115.85 %; the PM H+-ATPase activity and phospholipid contents increased slightly. This is consistent with the lower Na+ content and Na+/K+ ratio in leaves of salt-tolerant Jian 4 under salt stress.

Percoll density gradient-enriched populations of rat pituitary cells: Interleukin 6 secretion, proliferative activity, and nitric oxide synthase expression

We used a discontinuous Percoll density gradient centrifugation to prepare enriched populations of prolactin (PRL), growth hormone (GH), and folliculo-stellate (FS) cells from rat anterior pituitaries in order to characterize these various cell populations. After cell dissociation and centrifugation, enriched PRL cells (55% of total cells as determined by immunocytochemistry [ICC] were present in Fraction 1 (Fr1) (density ([d]) = 1.059). Fr2 (d=1.071) had enriched S100-positive FS cells (31% of total cells), but enriched GH cell (60% of total cells) were present in Fr3 (d=1.094). Interleukin 6 (IL-6) was secreted mainly by enriched PRL cells in Fr1 (350 pg/mL/106 cells) and Fr2 (194 pg/mL/106 cells), and much less by the enriched GH cells inFr3 (16 pg/mL/106). Proliferation studies with combined 3H-thymidine and ICC for pituitary hormones showed that only the PRL cell had significant prolifereative activity. Immunostaining showed that immediately after separation, all three isoforms of nitric oxide synthase (NOS) were expressed anterior pituitary cells. After 3 d of culture, there was a marked increase in nuclear staining for neuronal NOS (nNOS) in all three fractions, whereas inducible NOS (iNOS) and endothelial NOS (eNOS) rexpression did not change significantly. These results indicate that: 1. Enriched populations of PRL, FS, and GH pituitary cells can be readily obtained with a rapid discontinuous percoll density separation procedure. 2. PRL cells from different fractions of the gradient show differenet proliferation rates and IL-6 secretion varied in different enriched cell populations. 3. Although all three isoforms of NOS were expressed in rat pituitary cells, nNOS is the prindipal isoform in anterior pituitary cells, and its expression was icreased after 3 d of culture of anterior pitutuitary cells.

Interactions between lipopolysaccharides and blood factors on the stimulation of equine polymorphonuclear neutrophils

In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown. We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils. The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (≥99% neutrophils; viability ≥98%). Increasing concentrations of Escherichia coli ( E. coli) LPS (from 0.01–10 μg ml −1) were used to activate the neutrophils. When LPS was used directly, without another stimulator, the respiratory burst of neutrophils was not activated ( N=12 horses; n=5 assays per horse). On the contrary, when LPS was added to whole blood, the neutrophils isolated from this blood were stimulated in a LPS dose-dependent manner, but polymyxin B added to whole blood suppressed this stimulation ( N=2; n=6). LPS dissolved in autologous equine plasma stimulated the isolated neutrophils in a dose-dependent manner from 0.1–10 μg ml −1 ( N=5; n=12). Heat inactivation of the plasma abolished this CL increase ( N=2; n=5). LPS added to equine albumin did not stimulate the isolated neutrophils ( N=2; n=5). On the contrary, the addition of γ-globulins (1 mg ml −1) to LPS (10 μg ml −1) led to the stimulation of neutrophils ( N=2; n =5). We concluded that LPS did not directly stimulate the isolated equine neutrophils, but that plasmatic factors are needed for the stimulation of these cells by LPS.

Phosphatidylinositol 4-Phosphate Synthesis in Immunoisolated Caveolae-like Vesicles and Low Buoyant Density Non-caveolar Membranes

This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae-like vesicles, to produce PtdIns4P. The addition of recombinant phosphatidylinositol transfer protein increased PtdIns 4-kinase activity both in immunoisolated caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membrane rafts are the main site for cellular PtdIns4P production.