Percoll Gradient Centrifugation Research Articles

Proinflammatory Cytokines as an Intermediate Factor Enhancing Lipid Sperm Membrane Peroxidation in In Vitro Conditions

We have examined the effect of white blood cells (WBCs), various proinflammatory cytokines, or a combination of the two on the peroxidation of human sperm membrane lipids in in vitro conditions. Six recombinant cytokines, such as interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, IL-18, and tumor necrosis factor alpha (TNF-alpha), used singly or in combinations, were analyzed. WBCs were isolated from the whole heparinized blood using a density gradient technique (Histopaque 1.077). Spermatozoa were isolated from semen samples with normal sperm parameters by both the swim-up technique (swim-up fraction) and by a discontinuous Percoll gradient centrifugation (90% and 47% Percoll fractions). Peroxidative damage to sperm membrane lipids was assessed by determining the concentration of malondialdehyde (MDA) in lysates of spermatozoa using high-performance liquid chromatography (HPLC). There were no statistically significant differences in MDA concentrations between sperm fractions incubated with cytokines and respective controls (spermatozoa alone). In spermatozoa isolated by the swim-up technique, the MDA level was significantly higher only after incubation with IL-6 and IL-8 plus WBCs when compared to sperm incubated with leukocytes alone (0.62 +/- 0.21 micromol/L and 0.42 +/- 0.22 micromol/L, respectively; P < .05). In spermatozoa recovered from the 47% Percoll, only a combination of IL-12 and IL-18 used together with WBCs was linked with a significant increase in MDA concentration (from 0.41 +/- 0.13 micromol/L to 0.65 +/- 0.19 micromol/L; P < .05). The results obtained suggest that cytokines produced during the inflammatory process intensify the level of oxidative stress caused by leukocytes, which may have serious consequences for sperm membrane integrity.

Innate immune reactivity of the liver in rats fed a cholinedeficient L-amino-acid-defined diet

To investigate the innate immune reactivity of tumor necrosis factor-alpha (TNF-alpha), Toll-like receptor 4 (TLR4), and CD14 in the liver of non-alcoholic steatohepatitis (NASH) model rats. Male F344 rats were fed a choline-deficient L-amino-acid-defined (CDAA) diet. The rats were killed after 4 or 8 wk of the diet, and their livers were removed for immunohistochemical investigation and RNA extraction. The liver specimens were immunostained for TNF-alpha, TLR4, and CD14. The gene expressions of TNF-alpha, TLR4, and CD14 were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Kupffer cells were isolated from the liver by Percoll gradient centrifugation, and were then cultured to measure TNF-alpha production. The serum and liver levels of TNF-alpha in the CDAA-fed rats increased significantly as compared with the control group, as did the immunohistochemical values and gene expressions of TNF-alpha, TLR4, and CD14 with the progression of steatohepatitis. TNF-alpha production from the isolated Kupffer cells of the CDAA-fed rats was elevated by lipopolysaccharide stimulation. The expressions of TNF-alpha, TLR4, and CD14 increased in the NASH model, suggesting that TLR4 and CD14-mediated endotoxin liver damage may also occur in NASH.

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 x 10(6) cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 x 10(3) cells per well. Cells were grown for 1 day in MEMalpha plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 microM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.

Relationship between Insuline-like Growth Factor-I and Progesterone Secretion of Cultured Human Trophoblast Cells in Vitro

Objective To investigate the effect of insuline-like growth factor-I (IGF-I) on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradient centrifugation for primary culture. After stimulated with different concentrations(100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml) of IGF-I at the same time and with different duration(12 h, 24 h, 48 h, 72 h) of IGF-I with the same concentration, progesterone levels in the media were measured by radioimmunoassay. Simultaneously, semiquantitative reverse transcriptase polymerase chain reaction(RT-PCR) was applied to determine the expression of low density lipoprotein receptor (LDLR) mRNA. Results Progesterone levels correlated positively with IGF-I along with the IGF-I concentration increasing, progesterone level began to increase at 12 h, and reached the climax at 48 h when cultured with 100 µg/L IGF-I. The expression of LDLR mRNA was detectable in every group and accordant with variation of progesterone level. Conclusion Progesterone secretion has time- and dose-dependent effect on IGF-I, and IGF-I can up-regulate the expression of LDLR mRNA. IGF-I may play an important role in promoting secretion of progesterone in trophoblast cells.

Freezing-free preservation of human spermatozoa – a pilot study

This study tested a method for maintaining human spermatozoa without freezing for subsequent use in intracytoplasmic sperm injection (ICSI). We demonstrated that human sperm stored in electrolyte-free solution maintain their motility and viability for at least 4 and 6 weeks, respectively. We also have shown that preserved spermatozoa are fully functional in ICSI. Sperm chromosome analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage does not negatively affect sperm DNA integrity. A mouse model was used to analyze the ability of preserved sperm to participate in normal embryogenesis. Mouse sperm preserved in electrolyte-free solution in a similar manner as human sperm maintained motility for up to 3 weeks. When mouse spermatozoa stored for 1 week were injected into the oocytes by ICSI, they yielded normal blastoctysts and normal viable fetuses. The results of the study bear significance for human assisted reproduction technologies (ART) and provide clinicians and inf...

Neutrophil Elastase Converts Human Immature Dendritic Cells into Transforming Growth Factor-β1-Secreting Cells and Reduces Allostimulatory Ability

During microbial infection, neutrophils (polymorphonuclear leukocytes; PMNs) activate dendritic cells (DCs). However, early reports illustrated that neutrophil-derived mediators may suppress responses to mitogens. In the present study, we investigated the mechanism used by PMNs to modulate the immunostimulatory ability of DCs. Autologous syngeneic PMNs decreased T-cell proliferation induced by allogeneic DCs. Culture supernatant (CS) derived from PMNs also decreased allostimulation ability of immature DCs and increased the expression of transforming growth factor (TGF)-beta1 on DCs. A TGF-beta1 monoclonal antibody, a CD40 monoclonal antibody, or a serine protease inhibitor reversed the effect of PMN CS on DC allostimulatory ability. Furthermore, elastase reproduced the inhibitory effect of PMN CS on DC allostimulatory ability and the TGF-beta1 production. The role of elastase was confirmed by examining PMN CS from two patients with cyclic neutropenia, a disease due to mutations in the neutrophil elastase gene. These PMN CS samples had reduced elastase activity and were unable to increase DC TGF-beta1 production. Moreover, elastase and PMN CS induced IkappaBalpha degradation in DCs. We conclude that PMNs decrease DC allostimulatory ability via production of elastase leading to a switch of immature DCs into TGF-beta1-secreting cells.

Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

BackgroundPolymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.ResultsTo identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis.ConclusionOur data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

A study of the process of synchronisation and micronucleation in Beta vulgaris and the monitoring of an isolation procedure for micro-nuclei and micro-protoplasts by confocal laser scanning microscopy and flow cytometry

The process of synchronization and micro-nuclei induction in a suspension culture of Beta vulgaris, was induced by the sequential treatment with the DNA-synthesis inhibitor aphidicolin (30 μM, 24 h) and the spindle-toxin amiprophos-methyl (32 μM, 24 h). Mitotic arrest of divisions, spreading of G2-metaphase chromosomes, re-grouping of chromosomes, formation of a nuclear cell wall around single and re-grouped chromosomes and restitution of nuclei with a doubled DNA content was observed. The process of micro-nucleation was induced much less efficiently in Beta vulgaris than in Nicotiana plumbaginifolia. Cytological observations and monitoring of the process with flow cytometry and confocal laser scanning microscopy, was essential to follow up the course of events and to monitor the development of an efficient procedure for micro-protoplast isolation. Micro-nucleated protoplasts were fractioned by iso-osmotic Percoll gradient centrifugation to obtain heterogeneous micro-protoplast populations with cytoplasts and micro-protoplasts of different size. An enriched fraction with small sub-diploid micro-protoplasts was obtained with the equivalent DNA content of 1–4 chromosomes, as revealed by confocal laser scanning microscopy and flow cytometry. Sub-diploid micro-protoplasts with DNA amounts equivalent to 1–4 chromosomes were predominantly observed in the size classes of 1.8–10.2 μm at a frequency of 34.2–34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micro-nucleation followed the same course of cellular events as observed in N. plumbaginifolia. The correlation between DNA content and size of micro-nuclei and micro-protoplasts was not linear and affected by the degree of DNA condensation, total amount of DNA, and the presence of cytoplasm.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Identification of gene products present in Triton X‐100 soluble and insoluble fractions of human spermatozoa lysates using LC‐MS/MS analysis

A comprehensive analysis of the proteins found in human spermatozoa is essential for understanding the events leading up to, and including, fertilization and development. Proteomics offers a platform for investigating this process, provided that the dynamic range is relatively low. In this report, spermatozoa from a number of human sperm ejaculates were isolated in a pure state using discontinuous Percoll gradient centrifugation. Triton X-100 soluble and insoluble proteins were recovered and separated by SDS-PAGE. The separation lanes were dissected into 96 fractions and analyzed individually by LC-MS(n) . A comprehensive protocol, involving LC-MS/MS analysis eventually down to the ninth most intense peak found in the MS-survey scan, was performed. Analysis of purified human sperm populations resulted in the identification of 1056 gene products, of which approximately 8% have not previously been characterized. The data were supported by the large number of proteins represented by expressed sequence tags in the testis. Bioinformatic analysis demonstrated that 437 of the gene products were involved in various metabolic pathways including glycolysis and oxidative phosphorylation. The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual-oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa. Furthermore, a number of different classes of receptor have also been detected in these cells and are potential regulators of sperm function. This list includes at least six seven-pass transmembrane receptors, six tyrosine kinase receptors, a tyrosine phosphatase receptor, glutamate-gated ion channel receptors, transient receptor potential cation channels, and a non-genomic progesterone receptor. This is the first published list of identified proteins in human spermatozoa using LC-MS/MS analysis.

Effects of polychlorinated biphenyl (Aroclor 1254) on steroidogenesis and antioxidant system in cultured adult rat Leydig cells

Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental contaminants that disturb normal endocrine functions, including gonadal functions in humans and mammals. In the present study, we examined the direct effects of PCB on rat Leydig cells in vitro. Adult Leydig cells were purified by Percoll gradient centrifugation method and the purity of Leydig cells was also determined by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining method. Purified Leydig cells were exposed to different concentrations (10(- 10)-10(- 7) M) of PCB (Aroclor 1254) for 24 h under basal and LH-stimulated conditions. After the experimental period, cultured media were collected and used for the assay of testosterone and estradiol. The treated cells were used for the quantification of cell-surface LH receptors and activities of steroidogenic enzymes, such as cytochrome P(450) side-chain cleavage enzyme (P450scc), 3beta-HSD, and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Leydig cellular enzymatic antioxidants, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, gamma-glutamyl transpeptidase, glutathione-S-transferase, and nonenzymatic antioxidants, such as vitamins C and E, were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. In addition, total RNA was isolated from control and Aroclor 1254-exposed Leydig cells to monitor the steady-state mRNA levels by reverse transcription(RT)-PCR for steroidogenic acute-regulatory (StAR) protein, cytochrome P450scc, 3beta-HSD, and 17beta-HSD. Our results indicated that Aroclor 1254 (10(- 9), 10(- 8), and 10(- 7) M) treatments significantly inhibit basal and LH-stimulated testosterone and estradiol production. In addition, the activities of steroidogenic enzymes, enzymatic and nonenzymatic antioxidants were significantly diminished in a dose-dependent manner. However, LPO and ROS were elevated in a dose-dependent manner under basal and LH-stimulated conditions. RT-PCR analysis of StAR mRNA level showed a decrease only in 10(- 7) M dose of Aroclor 1254 treatment, while cytochrome P(450)scc, 3beta-HSD, and 17beta-HSD mRNAs were drastically decreased in both 10(- 8) and 10(- 7) M Aroclor 1254 treatment. These findings suggest that PCBs can act directly on Leydig cells to diminish testosterone production by inhibiting gene expression of steroidogenic enzymes and antioxidant system.

203 PRETREATMENT OF BOVINE SPERM OR OOCYTE WITH ANTIBODY TO LIPOCALIN-TYPE PROSTAGLANDIN D SYNTHASE INHIBITS FERTILIZATION

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatments: (1) no antibody (control), (2) α L-PGDS at fertilization time, (3) α L-PGDS-treated oocytes, or (4) α L-PGDS-treated sperm. Oocytes were inseminated with 10 � 104 washed spermatozoa in 4-well culture dishes. After 18 h (39�C, 5% CO2 in air), oocytes were vortexed to remove cumulus cells and accessory spermatozoa, and fixed in 3.7% paraformaldehyde and 10% Triton X–100 for 1 h. Oocytes were washed and transferred to a solution with PBS, 0.3% BSA, and 1% Triton for 1 h, stained with Hoechst 33342, and observed in the presence of 2 pronuclei in the cytoplasm. There were 4 replicates of 200 to 250 oocytes for fertilization assays. Data were analyzed by SAS. Addition of α L-PGDS with sperm, oocytes, or both significantly decreased fertilization (P &lt; 0.05) compared with the control: (1) 89.2 � 2.0%; (2) 19.4 � 2.0%; (3) 27.2 � 3.1%; or (4) 14.1 � 3.4%. These studies demonstrated that a rabbit polyclonal antibody against recombinant bovine L-PGDS reacts with both oocytes and spermatozoa, resulting in inhibition of fertilization in vitro, and has a possible role in bovine fertilization. This study was supported by FAPESP #2007/00363-5 and #2006/06008-0, Brazil.

A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii

This method has been developed to yield highly purified intact chloroplasts from Chlamydomonas reinhardtii. This procedure involves breaking cell-wall-deficient cells by passage through a narrow-bore syringe needle and purifying the intact chloroplasts by differential centrifugation and Percoll gradient centrifugation. This procedure can be completed in less than 3 h and is capable of generating relatively high yields of chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.

Advanced methods of isolation and identification of porcine primordial follicles

An effective isolation and identification method of primordial follicles would greatly benefit the animal production practice, transgenic animal production and endangered species conservation in the future. This study has not only advanced the isolation method but also developed an identification marker of primordial follicles. After enzymatic digestion, Percoll gradient centrifugation and mesh filtrations, the obtained follicular separations were then subjected to a cell sorter in order to collect primordial follicles. The study greatly improved the yield of primordial follicles (from about 1.85 × 10 5 to 7.79 × 10 5 per prepubertal ovary) by means of increasing cell layer number from 1 ml to 2.5 ml after Percoll gradient centrifugation. Based on traditional morphological criteria, the purity of recovered primordial follicles was averagely about 82.43 ± 9.41% ( n = 5) because of their similar size and appearance with somatic cells. To further exactly appreciate the purity of sorted primordial follicles, a germ cell-specific protein, MSY2, was used to recognize the oocytes of primordial follicles. The results of repeat experiments showed that about 98.31 ± 0.73% ( n = 4) of the primordial follicles cluster was MSY2-positive, which indicated the identification method of primordial follicles was more effective and high-yielded than the previous methods because of its higher purity.

Toxic effects of zearalenone and α-zearalenol on the regulation of steroidogenesis and testosterone production in mouse Leydig cells

Zearalenone (ZEA) and its derivative α-zearalenol (α-ZOL) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to animal fertility disturbances and other reproductive pathologies. The previous study demonstrated the toxic effects of ZEA and α-ZOL through disturbances in male fertility and other reproductive pathologies in mice. In this study, we further examined the direct biological effects of ZEA and α-ZOL on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine ZEA and α-ZOL-induced biological consequences, we measured testosterone secretion and transcription level of 3 key steroidogenic enzymes including 3β-HSD-1, P450scc and StAR, in ZEA and α-ZOL/human chorionicgonadotropin (hCG) co-treated cells. Our results showed that ZEA and α-ZOL (10 −4 M, 10 −6 M and 10 −8 M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The suppressive effect is correlated with a decrease in the level of transcription of 3β-HSD-1, P450scc, and StAR ( P < 0.05).

Immunophilin-like TWISTED DWARF1 Modulates Auxin Efflux Activities of Arabidopsis P-glycoproteins

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.

Simultaneous flow cytometric assessment for cellular types and phagocytic abilities of the haemocytes of the hard clam, Meretrix lusoria

The aim of this study was to devise a simple protocol for flow cytometric analysis to separate various haemocytic populations of the hard clam Meretrix lusoria based on the mitochondrial membrane potential diversity detected by the fluorescence probe 3,3-dihexyloxacarbocyanine iodide (DiOC6). Compared with the traditional technique for separation of haemocytic populations, continuous Percoll gradient centrifugation, our novel method was more efficient and yielded a higher ratio in separating the clams' haemocytic populations. Based on fluorescence 1 (FL-1) and side scatter (SSC) analysis for haemocytes stained with various fluorescent densities of DiOC6 using flow cytometer, the data showed that there were three obvious cell regions R1, R2, and R3, identified by hyalinocytes, small granulocytes and large granulocytes, respectively. At the same time our results showed that the percentages of haemocytes in R1, R2, and R3 were 49.71 ± 0.65%, 19.35 ± 00.74%, 30.94 ± 0.69%, respectively. After classifying the haemocytic populations, phagocytic activity of the haemocytes was simultaneously analysed with phycoerythrin (PE)-labeled Vibrio vulnificus and detected by flow cytometry. Our results demonstrated that there were higher percentages of large granulocytes compared with hyalinocytes and the percentage of small granulocytes was related to the mitochondrial membrane potential and phagocytic activities.

The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria

Characteristics of the succinate-supported H 2O 2 formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H 2O 2 generation measured in mitochondria prepared with digitonin (600.6 ± 26.8 pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H 2O 2 formation was significantly smaller in Percoll-purified mitochondria (252.6 ± 17.3 pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H 2O 2 formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185 ± 3.2 mV) and addition of BSA (0.025%) to the assay medium increased H 2O 2 generation by only 50%. In Percoll-purified mitochondria Δ Ψm was more depolarized (171 ± 2 mV) and BSA caused hyperpolarization by 10.7 ± 1.9 mV. H 2O 2 formation, which was largely independent of Δ Ψm, was stimulated by 400%, became highly dependent on Δ Ψm and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10 mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on Δ Ψm.

Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment

Heart attacks and congestive heart failure remain among the world's most prominent health challenges despite the many breakthroughs. Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into myocytes if an appropriate cardiac environment is provided. This study is meant to investigate the ability of BMSCs to differentiate into cardiomyocytes in a conditioned medium. BMSCs were isolated from rat femurs and tibias using Percoll gradient centrifugation method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. BMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semipermeable membrane. BMSCs marker of CD29 were highly expressed (98.89 ± 1.2%); however, CD34 could hardly be identified (5.61 ± 0.1%). After coculturing with myocytes, some of BMSCs showed contraction which became more regular and more vigorous. As assessed by RT-PCR, SERCA2 and RyR 2 were expressed by newly formed cells from 1 to 3 weeks. Immunostaining of newly differentiated BMSCs revealed positivity for cTnT. Some of these cells were positive for sarcomeric α-actinin, desmin, cTnT, and cTnI. Western blotting showed that cTnI protein expression was upregulated in these cells from 1 to 3 weeks. Newly formed BMSCs exhibited ultrastructural features of sarcomere formation and inward rectifier potassium current ( I K1). It is concluded that BMSCs possess the potential to differentiate into cardiomyocytes in the cardiac environment. BMSCs provide an excellent model for development of stem cell therapeutics, and their potential in the cardiac repair under various pathological conditions.

β-Adrenergic Receptor Stimulation and Adenoviral Overexpression of Superoxide Dismutase Prevent the Hypoxia-mediated Decrease in Na,K-ATPase and Alveolar Fluid Reabsorption

Hypoxia has been shown to cause lung edema and impair lung edema clearance. In the present study, we exposed isolated rat lungs to pO(2) = 40 mm Hg for 60 min or rats to 8% O(2) for up to 24 h and then measured changes in alveolar fluid reabsorption (AFR) and Na,K-ATPase function. Low levels of oxygen severely impaired AFR in both ex vivo and in vivo models. The decrease in AFR was associated with a decrease in Na,K-ATPase activity and protein abundance in the basolateral membranes from peripheral lung tissue of hypoxic rats. Beta-adrenergic agonists restored AFR in rats exposed to 8% O(2) (from 0.02 +/- 0.07 ml/h to 0.59 +/- 0.03 ml/h), which was associated with parallel increases in Na,K-ATPase protein abundance in the basolateral membrane. Hypoxia is associated with increased production of reactive oxygen species. Therefore, we examined whether overexpression of SOD2, manganese superoxide dismutase, would prevent the hypoxia-mediated decrease in AFR. Spontaneously breathing rats were infected with a replication-deficient human type 5 adenovirus containing cDNA for SOD2. An otherwise identical virus that contained no cDNA was used as a control (Adnull). Hypoxic Adnull rats had decreased rates of AFR (0.12 +/- 0.1 ml/h) as compared with hypoxic AdSOD2 and normoxic control rats (0.47 +/- 0.04 ml/h and 0.49 +/- 0.02 ml/h, respectively), with parallel changes in Na,K-ATPase.