Percoll Density Gradient Centrifugation Research Articles

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.

Effects of arecoline on testosterone release in rats

Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 microg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentrations of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34 degrees C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10(-8) to 10(-6) M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10(-6) M), or 8-Br-cAMP (10(-5) M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17beta-hydroxysteroid dehydrogenase and enhancing the expression of StAR.

Loss of Pten, a tumor suppressor, causes the strong inhibition of autophagy without affecting LC3 lipidation

1Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [14C]-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microsopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.

Inhibitory Effects of Oral Administration of Nonylphenol on Testosterone Production in Rat Leydig Cells.

Nonylphenol (NP) is the final metabolite of nonyphenol polyethoxylate (NPE), a non-ionic surfactant that is frenquently incorporated into detergent and pesticide formulation. This metabolite has a structure mimicking 17β-estradiol and has been reported to have xenoestrogenic effects, and may affect the endocrine system. Its effects on testosterone release from Leydig cells are unclear. In the present study, we investigated the influence of NP, administered orally (gavage) or delivered via spray on Leydig cell activity and testosterone release. Rat Leydig cells were prepared by enzymatic dispersion and then percoll density gradient centrifugation. The purified Leydig cells (1×105 cells/ml) were incubated with or without human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (FSK, an adenylyl cyclase activator, 10-5 M), A23187 (a calcium ionophore, 10-5 M), androstenedione (a precursor of testosterone, 10-6 M), or 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP, a cell-permeable cAMP analogue, 10-5 M) at 34°C for 1 hour. Testosterone levels in the media were measured by radioimmunoassay. The data showed that the administration of NP via spray did not affect steroidogenesis of testosterone in rat Leydig cells. In contrast, oral administration of NP (100 mg/kg) once per day for 30 days had inhibitory effect on steroidogenesis of testosterone in rat Leydig cells. In the in vivo experiments, administration of NP by oral ingestion decreased plasma testosterone concentrations in rats. Furthermore, oral NP induced a significant inhibition on the release of testosterone in vitro in response to hCG, 8-Br-cAMP, and FSK. Moreover, administration of NP also caused a decrease on testosterone release in response to androstenedione and A23187. The in vitro experiments revealed that oral NP at a dose of 100 mg/kg/day suppressed testosterone biosynthesis by inhibiting the 17β-hydroxysteroid dehydrogenase activity, cyclic AMP pathway and calcium influx in Leydig cells. These results suggested that oral administration of NP had inhibitory effects on steroidogenesis of testosterone in rat Leydig cells and might provide more information about the toxic effects of nonylphenol on reproductive endocrine disrupt.

Cultured Bovine Trophoblast Cells Differentially Express Genes Encoding Key Steroid Synthesis Enzymes

Placental trophoblasts are an important source of endocrine, paracrine and autocrine acting hormones. The aim of the present study was to establish and evaluate a tissue culture model for bovine trophoblasts to study regulation of key genes of steroid hormone synthesis. Trophoblast cells were isolated from cotyledons by collagenase disaggregation and subsequent percoll density gradient centrifugation. The cells were seeded on collagen coated dishes and incubated for up to seven days. The cells were characterized for the presence of mesenchymal vimentin and epithelial cytokeratin filaments and for Dolichos biflorus agglutinin (DBA) binding, a marker for differentiated trophoblast giant cells. Transcripts of Hsd3b, Cyp17 and Cyp19 encoding 3β-HSD, P450c17 and P450arom, the key enzymes of progesterone, androgen, and oestrogen biosynthesis, respectively, and of Csh1 encoding the trophoblast-specific hormone placental lactogen (PL) were measured by qPCR. Uninucleate cotyledonary epithelial cells and bi- and trinucleate trophoblast giant cells efficiently formed a dense cell layer on the collagen coated dishes within 24 h. Bi- and trinucleate cells showed DBA binding and weak or undetectable cytokeratin immunoreactivity. Vimentin-positive, fibroblast-like cells were found on top of this cell layer. Cyp19 transcripts were found in freshly dissociated but not in cultured cells. Cyp17 expression continuously increased, Hsd3b transcripts largely and rapidly increased during the first days in culture, followed by a decline after three days, whereas Csh1 decreased towards day seven. Serum free culture conditions significantly enhanced Cyp17 and Csh1 but not Hsd3b expression. The data indicate that collagen is a favourable substrate for cultured binucleate trophoblast giant cells. The cells represent an in vitro model to study the regulation of key genes of placental progesterone and androgen but not of oestrogen biosynthesis.

Proteomic Analysis of Highly Purified Peroxisomes from Etiolated Soybean Cotyledons

To identify previously unknown peroxisomal proteins, we established an optimized method for isolating highly purified peroxisomes from etiolated soybean cotyledons using Percoll density gradient centrifugation followed by iodixanol density gradient centrifugation. Proteins in highly purified peroxisomes were separated by two-dimensional PAGE. We performed peptide mass fingerprinting of proteins separated in the gel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and used the peptide mass fingerprints to search a non-redundant soybean expressed sequence tag database. We succeeded in assigning 92 proteins to 70 sequences in the database. Among them, proteins encoded by 30 sequences were judged to be located in peroxisomes. These included enzymes for fatty acid beta-oxidation, the glyoxylate cycle, photorespiratory glycolate metabolism, stress response and metabolite transport. We also show experimental evidence that plant peroxisomes contain a short-chain dehydrogenase/reductase family protein, enoyl-CoA hydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-like protein and a voltage-dependent anion-selective channel protein.

Expression of the two‐pore domain potassium channel, TASK‐2, in rat pancreatic acinar cell zymogen granule membrane

Ion flux across pancreatic secretory vesicle membranes contributes to exocytotic secretion: mediating vesicular swelling involved in “kiss and run” exocytosis, promoting solubilization and release of vesicle content, and contributing to intravesicular pH changes. The present study implicates the pH sensitive TASK‐2 channel in one or more of these processes: TASK‐2 expression was documented and localized to zymogen granule membranes and secretion shown to be modulated by TASK‐2 selective drugs. TASK‐2 mRNA expression was demonstrated using SYBR Green real time PCR. Total RNA was isolated from purified acinar cells obtained by Percoll density gradient centrifugation of collagenase‐dispersed cells. Reverse transcription was performed on 1 μg of DNase‐treated total RNA using random decamer primers. TASK‐2‐specific forward and reverse primers amplified a single product from the cDNA based on dissociation curve analysis. Western blot analysis using an anti‐TASK‐2 antibody (Alomone) revealed two immunoreactive protein bands in a crude microsomal fraction migrating as a doublet at 54 and 55 kD (the predicted mass of rTASK‐2 is 58 kD); identical protein bands were obtained in Western blots of purified zymogen granule membranes. Confocal microscopy revealed that immunoreactivity was cytoplasmic and primarily localized to the apical pole. Finally, CCK‐induced amylase secretion from diced pancreatic tissue fragments was increased 100% by the TASK‐2 agonist, halothane, without affecting basal release and completely inhibited by the TASK‐2 inhibitor, clofilium, without affecting basal secretion.

RELEASE OF THE LIPID PEROXIDATION MARKER 8-EPI-PROSTAGLANDIN F2α FROM ISOLATED GILL PAVEMENT CELLS

The aim of the present study was to evaluate oxidative injury in gill pavement cells (GPCs) from fathead minnow (Pimephales promelas) using F2-isoprostane (F2-iP) release as an index of lipid peroxidation. Cells were isolated from pooled gill tissue by collagenase treatment, mechanical sieving, and Percoll density gradient centrifugation. Baseline levels of 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha) were measured by incubating GPCs in physiological buffer (10(6) cells/ml) and enzyme immunoassay. After 60 min, the amount of immunoreactive 8-epi-PGF2 alpha (ir8-epi-PGF2 alpha) in control medium ranged from 1,374 to 5,515 pg/ml. Lead nitrate, 0.6 to 120 microM, did not influence ir8-epi-PGF2 alpha release, whereas FeCl3 stimulated release at 500 microM but not at 5 microM. Incubation medium was extracted for acidic lipids and analyzed by liquid chromatography/mass spectrometry/electrospray ionization. A compound in the medium exhibited a retention time on reverse-phase high-performance liquid chromatography nearly identical to that of synthetic 8-epi-PGF2 alpha The mass spectrum taken from the total ion chromatogram from 14.8 to 15.1 min contained a prominent ion at m/z 353, as expected for the molecular ion of 8-epi-PGF2 alpha. Similar results were obtained with tissue subjected to base hydrolysis. Mass spectra of extracted ion chromatograms obtained with gill extracts and authentic standard showed a close correspondence of fragment ions, providing definitive evidence for production and storage of F2-iPs by fish gills. In summary, F2-iP release occurs during lipid peroxidation injury to fish gill epithelium, and its measurement may facilitate aquatic toxicology studies of metallic and nonmetallic contaminants.

Essential Role of Monocytes in theIn VitroProduction of IL-4 and Nonspecific IgE Antibody by Peripheral Blood Lymphocytes from Mice Sensitized s.c. Once with Cedar Pollen

To explore which cytokine or cell is essential for the production of antibodies (Abs) of the IgE class in allergic diseases, we injected cedar pollen into wild-type, interferon-gamma(-/-) (IFN-gamma(/)), or interleukin-4(-/-) (IL-4(-/-)) BALB/c mice through four (i.n., i.p., s.c., and i.v.) different routes without adjuvant. Wild-type or IFN-gamma(-/-), but not IL-4(-/-), mice sensitized once or twice showed a significant increase in total IgE Ab in their serum, revealing the essential role of IL-4 in the production of total IgE Ab. We separated peripheral blood mononuclear cells (PBMCs) from untreated or sensitized mice into monocyte-rich, lymphocyte-rich, and granulocyterich populations by Percoll density-gradient centrifugation or into specific antigen cells by flow cytometry, cultured the cells in various combinations, and examined the levels of cytokines and IgE Ab released into the medium. The PBMCs from mice sensitized s.c. once, but not those from untreated animals, produced significant amounts of IL-4 and total IgE Ab, whereas the lymphocyte-rich population alone did not. Unexpectedly, IL-4 and IgE Ab production was restored by the addition of Mac-1(+) cells in the monocyte-rich fraction to the lymphocyte-rich fraction. These results indicate the essential role of monocytes in the production of IL-4 and total IgE Ab by lymphocytes during the initial stage of sensitization.

Combined process of urea nitrogen removal in anaerobic Anammox co-culture reactor

In this study, the feasibility of biological urea nitrogen removal in anaerobic Anammox co-culture was investigated. After 100 days of operation, complete urea nitrogen removal of 0.35 g (NH 2) 2CO–N L −1 d −1 was achieved. The pure Anammox bacteria were obtained by percoll density-gradient centrifugation and found to be of incapable to hydrolyze urea. The ureolytic bacteria were isolated from the Anammox co-culture by the spread plate and streak. Comparative analysis of partial 16S rDNA sequence presented it belongs to Bacillus sp., and so named as Bacillus sp. LST-1. Fluorescence in situ hybridization was applied to identify the ratio of Bacillus sp. and Anammox in the reactor and the value was approximately 1:4. Urea nitrogen removal was realized in this autotrophic, anoxic reactor via the combined process of urea hydrolysis by Bacillus sp. LST-1 and ammonium oxidizing by Anammox. The investigation of this combined process might have an actual significance in engineering application for its low operational cost.

Mikrokalorimetrische Untersuchungen an bovinen Spermatozoen nach Percoll-Dichtegradienten-Zentrifugation - ein experimenteller Beitrag zum Einsatz in der Andrologie

Microcalorimetric measurements can be used for recording exothermic or endothermic summation effects of a great variety of biological processes. The aim of the present study was to examine the usefullness of the microcalorimetry method to characterise the biological activity of spermatozoa. The heat flow of bovine fresh sperm as well as cryosperm samples were measured after Percoll density-gradient centrifugation in a 4-channel microcalorimeter. Various calibration times, volumes of samples and sperm concentrations were tested and analysed. Sperm concentration was recorded by a computer-assisted, computer-aided software system method (CASA). Using a calibration time of 15 minutes, the heat signal of the fresh and cryosperm samples showed a characteristic peak after 39.5 min and 38.1 min (mean), respectively, with a significant correlation to sample volume and sperm concentration (p < 0.05). For obtaining the best results, a sample volume of 1 ml and a sperm concentration of more than 50 x 10 (6)/mL was used. With microcalorimetric measurements the biological activity of spermatozoa could be recorded for reproducible results, thus opening the way to an automatised ejaculate analysis in the future. More investigations are necessary to correlate microcalorimetric parameters with semen function.

PENINGKATAN KUALITAS SPERMATOZOA PADA PROSES PEMISAHAN SPERMATOZOA DENGAN SENTRIFUGASI GRADIEN DENSITAS PERCOLL MELALUI PEMBERIAN FOSFOLIPID

The separation of X and Y spermatozoa can be conducted by using Percoll Gradient Density Centrifugation(SGDP) method. It is, easy, valid, cheap and applicable method for developing made-insemination. Although it  has often been applied in sexing the spermatozoa, it was reported  that the method had often caused damages on the spermatozoa membranes and resulted in decreasing of the spermatozoa quality. The damages of spermatozoa after SGDP  process were specifically caused by (I) the loss of seminalplasma, (II) the increase of free radicals, and (III) the collision or friction among the spermatozoa. Among the three causal factors above, the third or the physical factor is claimed to be the most important one. It was assumed that the first and the second factors can be overcome if the third one is avoided. The spermatozoa membranes consist of lipids, protein, carbohydrate, and some substances at a low rate. Thegeneral objectives of this research was to identify the influence of the phospholipids PC (Phosphatidylcholin)to spermatozoa in order to avoid the damages of the spermatozoa in the process of SGDP.

Isolation, propagation and characterization of primary tubule cell culture from human kidney (Methods in Renal Research)

Proximal tubule cells (PTC) are the major cell type in the cortical tubulointerstitium. Because PTC play a central role in tubulointerstitial pathophysiology, it is essential to prepare pure PTC from kidney tissue to explore the mechanisms of tubulointerstitial pathology. The authors have successfully refined and characterized primary cultures of human PTC using Percoll density gradient centrifugation as a key PTC enrichment step. The cells obtained by this method retain morphological and functional properties of PTC and are minimally contaminated by other renal cells. In particular, the primary isolates have characteristics of epithelial cells with uniform polarized morphology, tight junction and well-formed apical microvilli. Cytokeratin is uniformly and strongly expressed in the isolates. Brush border enzyme activities and PTC transport properties are retained in the isolates. This method therefore provides an excellent in vitro model for the physiologic study of the human proximal tubule.

Distribution of necrotizing hepatopancreatitis bacterium (NHPB) in cultured white shrimp, Litopenaeus vannamei, from Mexico

This paper describes the first systematic monitoring conducted in 2002 at shrimp farms in Sinaloa and Sonora (Mexico) for the presumptive and confirmative diagnosis of necrotizing hepatopancreatitis bacterium (NHPB) in Litopenaeus vannamei. Light microscopy (wet mounts and hematoxilin-eosin-stained sections of hepatopancreas) showed atrophy of the hepatopancreas, strangulation and necrosis of the hepatopancreatic tubules, with masses of NHPB within the tubular epithelium, hemocytic infiltrates and tubular melanization. The gene encoding the 16S rRNA (16S rDNA) of NHPB was amplified by polymerase chain reaction (PCR) from clinical specimens and the bacteria isolated using Percoll density gradient centrifugation. Negative-stain transmission electron microscopy revealed pleomorphic bacteria, most of them ovoid or rod-shaped (0.2 µm wide and 0.6–0.9 µm long), and a helical form demonstrating seven to twelve spiral turns (0.23 µm wide and 2.43–5.27 µm long). Several severe epizootics of commercially grown L. vannamei occurred during the study period and the mortality was attributed to NHPB infection. Using PCR and microbiological analyses, other etiologic agents, such as white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, hepatopancreatic parvovirus and vibriosis, were ruled out as contributory factors.

Association of CD38 with Nonmuscle Myosin Heavy Chain IIA and Lck Is Essential for the Internalization and Activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.

Sperm Preparation: DNA Damage by Comet Assay in Normo- and Teratozoospermics

The present study was carried out on semen samples of human fertile and infertile subjects, teratozoospermics (TZs) and idiopathics (IDs), with neat semen and sperm prepared by swim up or Percoll density gradient centrifugation procedures. Sperm morphology analysis revealed that only head and midpiece defects in TZs and IDs were significantly (P < 0.001) higher compared to fertile subjects. Infertile subjects indicated significantly higher (P < 0.001) sperm DNA damage compared to fertile subjects. Fertile subjects with sperm prepared from neat and Percoll density gradient centrifugation exhibited a comet tail DNA percentage of 20% and 15%, respectively. The TZs and IDs infertile subjects had higher levels of comet tail DNA of 33% and 25% and 25% and 19%, respectively. A significant (F = 24.01; P = 0.0059) decrease in mean comet head DNA percentage or sperm DNA integrity was observed in neat samples from fertile and infertile subjects by Repeated Measures ANOVA. In Percoll prepared samples from fertile, TZs, and IDs, there was a significant increase in sperm DNA integrity. Similarily, there was a decrease in abnormal sperm morphology in swim up and Percoll prepared sperm compared to neat samples. The Percoll density gradient centrifugation procedure yields sperm with an increase in sperm DNA integrity relative to swim up. Sperm DNA damage of TZs with both sperm preparation methods was significantly (P < 0.01) higher when compared to fertile and IDs. But the level of DNA damage was higher in IDs compared to fertile subjects. Compared to the other methods tested, the Percoll method yielded sperm with improved DNA integrity. In conjunction with semen analysis, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating the patients to specific assisted reproductive treatments.

7 EFFECTS OF DIFFERENT TRYPSIN SOURCES AND CONCENTRATIONS ON THE VIABILITY OF BOVINE SPERM PRE- AND POST-CRYOPRESERVATION

The goal of this research was to investigate the effect of different sources and concentrations of trypsin on the viability of bovine sperm as a potential method for removing pathogens similar to the washing methods developed for embryos. Trypsin derived from porcine pancreas (Sigma-Aldrich, St Louis, MO, USA) at 2.5% and 0.25% was compared to the recombinant human sequence (TrypLE Select; Invitrogen, Carlsbad, CA, USA) at 10× and 1× concentrations. Cryopreserved bovine sperm (n = 3 bulls) were thawed and processed using discontinuous (90/45) Percoll density gradient centrifugation; the sperm pellets were then washed (10 min at 300g) in TL-HEPES Solution (Cambrex Corp., East Rutherford, NJ, USA) and then resuspended in 1 mL of the same medium. Aliquots of 200 µL of the washed sperm were then added to 1 mL of each of the 4 trypsin treatments as well as a negative control (without trypsin) and incubated at room temperature. Aliquots (25 µL) of each treatment were examined for progressive motility after 5 min. As a result, the control sperm (no trypsin) increased progressive motility by 6.7% and the 1× TrypLE treatment by 9.3%. However, the 10× TrypLE Select and the 10× and 1× porcine pancreas extracts decreased progressive motility by 8.3, 29.0, and 4.0%, respectively. The objective of the second experiment was to determine if the treatment of bovine semen with trypsin (1× TrypLE Select and 0.25% porcine pancreas extract) before or after cryopreservation would affect sperm quality as compared to cryopreservation without trypsin treatment. Raw semen (n = 6 bulls) was collected, evaluated, cryopreserved, and then thawed using a standard bovine method (Biladyl®; Minitube, Verona, WI, USA) without further treatment (control) or after treatment with one of two trypsin treatments (density gradient centrifugation with 1× TrypLE Select or 0.25% porcine pancreas extract in the 45% Percoll layer and a soybean trypsin in activator (Sigma) in the 90% layer) either before freezing (Treat–Freeze) or after thawing (Freeze–Treat). The results for the 6 individual bull samples were comparable and are presented as means (± SEM) compared to the cryopreserved control (no trypsin treatment). Using the Mann–Whitney Rank Sum Test, no differences (P &amp;gt; 0.05) were found in any of the parameters comparing the crypreserved controls (no trypsin treatment) and the 4 treatments: Freeze–Treat vs. Treat–Freeze using either the recombinant TrypLE Select (1×) or the porcine pancreas extract (0.25%). These results suggest that trypsin treatment, before or after cryopreservation, can be used safely on bovine sperm without affecting viability in vitro. Table 1.Comparison of cryopreserved bovine semen without and with various treatments

Mitochondria and apicoplast of Plasmodium falciparum: Behaviour on subcellular fractionation and the implication

The mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp. is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques – Percoll density gradient centrifugation and fluorescence-activated organelle sorting – for improving the purity of mitochondria isolated from the crude organelle preparation of Plasmodium falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium.

Comparative Study of Four Different Sperm Washing Methods Using Apoptotic Markers in Ram Spermatozoa

The accurate measurement of semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. A good sperm preparation technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. In this study, we investigate the effects of 4 sperm preparation techniques (a dextran/swim-up procedure, discontinuous Percoll density gradient centrifugation, sucrose washing, and filtration) on ram sperm quality parameters. Besides the evaluation of viability and the capacitation state, we also analyzed the apoptotic status of the sperm samples by assessing the phosphatidylserine translocation and caspase-3 and -7 activities. This is the first report, to our knowledge, that evidences the presence of active caspases in ram sperm. The results confirm the better ability of the dextran/swim-up procedure to select nonapoptotic spermatozoa, in addition to viable and noncapacitated sperm, compared with other sperm preparation methods. This should be considered to enhance results of artificial insemination techniques in ovine reproduction.