Resuspension In Medium Research Articles

Formation of Lutein, β-Carotene and Astaxanthin in a Coelastrella sp. Isolate.

In this study, the effect of media composition, N/P ratio and cultivation strategy on the formation of carotenoids in a Coelastrella sp. isolate was investigated. A two-stage process utilizing different media in the vegetative stage, with subsequent re-suspension in medium without nitrate, was employed to enhance the formation of carotenoids. The optimal growth and carotenoid content (β-carotene and lutein) in the vegetative phase were obtained by cultivation in M-8 and BG11 media. Use of a N/P ratio of 37.5 and low light intensity of 40 μmol m−2 s−1 (control conditions) led to optimal biomass production of up to 1.31 g L−1. Low concentrations of astaxanthin (maximum of 0.31 wt. %) were accumulated under stress conditions (nitrogen-deficient medium containing 1.5 % of NaCl and light intensity of 500 μmol m−2 s−1), while β-carotene and lutein (combined maximum of 2.12 wt. %) were produced under non-stress conditions. Lipid analysis revealed that palmitic (C16:0) and oleic (C18:1) constituted the main algal fatty acid chains (50.2 ± 2.1% of the total fatty acids), while esterifiable lipids constituted 17.2 ± 0.5% of the biomass by weight. These results suggest that Coelastrella sp. could also be a promising feedstock for biodiesel production.

Media Screening for Obtaining Haematococcus pluvialis Red Motile Macrozooids Rich in Astaxanthin and Fatty Acids

Astaxanthin from Haematococcus pluvialis is commercially produced in a two-stage process, involving green vegetative (macrozooid) and red aplanospore stages. This approach has been scaled up to an industrial process but constraints limit its commercial success and profitability, including: contamination issues, high pigment extraction costs, requirements for high light levels and photo-bleaching in the red stage. However, in addition to the aplanospore stage, this alga can produce astaxanthin in vegetative palmelloid and motile macrozooid cells. In this study, a two-stage process utilising different media in the green stage, with subsequent re-suspension in medium without nitrate was employed to optimise the formation of red motile macrozooids. Optimal growth in the green phase was obtained on cultivation under mixotrophic conditions in EG:JM media followed by re-suspension in medium without nitrate resulting in red motile macrozooids with an astaxanthin content of 2.74% (78.4% of total carotenoids) and a lipid content of 35.3% (rich in unsaturated fatty acids. It is envisaged that the red motile macrozooids could be harvested and fed as a whole-cell product directly in the animal feed and aquaculture sectors, or used as a blend of carotenoids and polyunsaturated fatty acids (PUFAs) in nutraceutical products.

Involvement of mitochondrial dysfunction in the adverse effect exerted by seminal plasma from men with spinal cord injury on sperm motility

The aetiology of severe asthenozoospermia in men with spinal cord injury includes an adverse impact of seminal plasma (SP) on sperm motility. In this study we investigated the effect exerted by SP from men with SCI on donor sperm mitochondrial activity and its reflection on motility. Donor spermatozoa were exposed (1h) to SP from 22 ejaculates of men with SCI. Only SP from samples exhibiting both a low fructose level and an inhibitory effect on mitochondrial membrane potential (ΔΨm), assessed at flow cytometry with JC-1, affected donor sperm motility when evaluated 1h after co-incubation. This effect was reverted by washing from SP and sperm re-suspension in medium containing glucose, in spite of persistently depressed ΔΨm. In the same samples, sperm motility and vitality dramatically decreased when evaluated 6h after washing and re-suspension in the glucose-containing medium. Seminal plasmas which induced a disruption of ΔΨm, also enhanced a mitochondrial ROS generation, as assessed by MitoSOX red. The enhanced mitochondrial ROS generation was associated with a late induction of sperm membrane lipid peroxidation, as assessed by BODIPY C11 , when evaluated at 6h, but not at 1h, after washing from SP. Furthermore, activation of caspase-9 and caspase-3 accompanied the loss of ΔΨm. In conclusion, a double energetic blockage (glycolysis and mitochondrial respiration) can represent a metabolic determinant of the early adverse effect exerted by SP from men with SCI on sperm motility. Mitochondrial dysfunction-related oxidative/apoptotic mechanisms can account for later consequences on sperm motility/vitality.

Polymer model of cancer cell adhesion to glycosaminoglycan substrates using the radius of gyration

AbstractThe hypothesis that the adhesion of whole cancer cells to glycosaminoglycan (GAG) substrates is a function of polysaccharide radius of gyration is presented. We use a worm‐like chain (WLC) polymer model describing the global structure of the GAGs that will take into account the charge distribution and contour length of the polysaccharide implicitly in its radius of gyration to relate these parameters to cell adhesion. Specifically, we present measurements of the in vitro adhesion of cancer cells to isolated and individualized GAG substrates. We find that adhesion of the cells has a linear response with the radius of gyration and is essentially controlled by the charge per dimer. This dominating mechanism is not eliminated when the cells are subjected to resuspension in media with heparin. We then propose how these physical properties could be used to predict the preferred molecular structures of compounds for use as antimetastatic or antiinflammatory agents by comparing our results with known effective molecules. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Bicarbonate and bovine serum albumin reversibly ‘switch’ capacitation-induced events in human spermatozoa

We have investigated the reversibility of biochemical and physiological changes that occur upon suspension of ejaculated human spermatozoa during in vitro capacitation. Cells were swum up in a simple HEPES-based saline [lacking bicarbonate and bovine serum albumin (BSA)], then resuspended either in supplemented Earle's balanced salt solution (sEBSS) (25 mM bicarbonate) with 0.3% BSA (for in vitro capacitation) or in medium-lacking bicarbonate and/or BSA. Progesterone-induced acrosome reaction (AR) developed during in vitro capacitation (6 h). A progesterone-induced [Ca2+]i signal was detectable in cells maintained in the simple HEPES-based saline, but upon transfer to sEBSS, the response increased three- to four-fold, saturating within <30 min. Serine/threonine phosphorylation saturated within minutes of resuspension, but tyrosine phosphorylation developed over 3 h. Return of cells to non-capacitating conditions caused reversal of all capacitation-dependent changes. The [Ca2+]i signal reverted to its 'uncapacitated' size within <30 min. Protein phosphorylation reversed gradually and could be reinduced (kinetics resembling the first response) upon resuspension in sEBSS. The ability of cells to undergo progesterone-induced AR fell to levels similar to those in uncapacitated cells within 1 h of resuspension in medium not supporting capacitation. Loss of protein phosphorylation occurred only in the absence of both bicarbonate and BSA, but effects on [Ca2+]i signalling and AR could be seen after removal of only one of these factors. We conclude that key events in the capacitation of human spermatozoa are both reversible and repeatable.

PRESERVATION OF EMBRYONIC KIDNEYS FOR TRANSPLANTATION

P30 Aims: Long-term storage of embryonic kidneys is crucial for the organisation of transplantation and organ banking. To date, ex vivo preservation of rat embryonic kidneys has proved feasible for three days in ice cold University of Wisconsin solution with added growth factors. In this present study, the effects of controlled rate freezing and ice-free vitrification on metanephroi (mn) viability were investigated. Vitrification is amorphous solidification of a supercooled liquid and it is achieved by adjusting the solute composition and the cooling rate such that nucleation and growth of ice crystals is prevented. Vitrification typically employs high concentrations of cryoprotectants to prevent ice formation with the risk of cryoprotectant-induced cytotoxicity. Methods: Metanephroi were isolated from 15-day (E15) timed pregnant Lewis rats, these were then randomly divided between three groups: control (C), controlled rate freezing (CRF) and vitrification (V). CRF metanephroi were cryopreserved using a DMSO/FCS/RPMI solution and controlled rate freezing at –0.3°C/min to -40°C prior to storage at -135°C for 48 hours. Mn were re-warmed by agitation in a 37°C waterbath until thawed, cryoprotectant was them removed and mn washed prior to re-suspension in media and subsequent viability analysis. Vitrified samples were cooled to -100°C in cryoprotectant (VS55, consisting of an 8.4M mixture of DMSO, formamide, and propanediol in EuroCollins solution) using an isopentane bath, then vitrified to -120°C and stored for 48 hours at -135°C. After storage mn were thawed to -35°C in a 30% DMSO bath then the cryoprotectant was diluted out by addition of mannitol in Unisol. The supernatant was removed and mn were resuspended in media prior to viability analysis. Mn viability was analysed for cell viability via an AlamarBlue assay, histology (light microscopy, T.E.M. and cryosubstitution) and transplantation to adult Lewis recipients. Results: Following normalisation, there was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved mn group relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing resulting in ‘scattered’ tissue and water inside the cells post-thawing. In contrast, the amount of ice was significantly reduced by vitrification. This was supported by light microscopy demonstrating significant vacuolation of the cytoplasm of control-rate frozen metanephroi following re-warming. By comparison, the re-warmed vitrified metanephroi had little cytoplasmic disruption. However, T.E.M demonstrated mitochondrial and nuclear injury at the cellular level. Transplantation of the mn is the ultimate test of viability, statistically less CRF metanephroi grew compared with control and vitrified metanephroi (p<0.05). Conclusions: There is a need for long-term storage of organs to make mn transplantation a realistic possibility. This study demonstrates that standard control-rate freezing methods are unsuitable for this purpose. Vitrification of mn using a protocol developed for blood vessels yielded more promising results but further development of the vitrification solution, exposure time and loading steps are required to reduce the observed toxicity and subsequent reduced viability.

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.

Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo‐3. inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: Tyrphostin A48, pertussis toxin, and 3‐quinuclidinyl benzilate

The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25 degrees C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]i. Initial [Ca2+]i was 231 +/- 58 nM (+/- SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca2+]i by 106 +/- 19 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 +/- 18 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca2+]i induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB), an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121-130). This [Ca2+]i increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca2+]i, all three inhibitors also blocked the zona pellucida-induced acrosome reaction. These results indicate that [Ca2+]i increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca2+]i suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding.

Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions.

We have cloned two metal-regulated genes (mrgA and mrgC) from Bacillus subtilis by using transposon Tn917-lacZ. Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct. Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper. Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription. Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions. Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression. We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC. Both genes are preceded by a predicted sigma A-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur). Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium.

Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida

The authors report a method of micromanipulation for the insertion of a single spermatozoon under the zona pellucida of human oocytes that results in a high rate of fertilization without damage to the oocyte. Spermatozoa were exposed to calcium-depleted medium containing strontium chloride for 20 to 24 hours before resuspension in medium containing calcium to induce capacitation. Single spermatozoa treated in this way were injected under the zona pellucida of morphologically mature oocytes and fertilization was confirmed by electron-microscopy. Five of seven oocytes manipulated within 9 hours of aspiration from the follicle and 3 of 12 oocytes manipulated 23 to 28 hours after recovery from the follicle fertilized. This technique has profound implications for the possible treatment of severe male infertility.

Studies on the osmotic disruption and resealing of synaptosomes.

The release of lactate dehydrogenase and K+ when synaptosomes are exposed to resuspension in media of various osmolarity has been investigated in order to measure their disruption. Even when resuspended in distilled water a significant percentage (10-20%) of lactate dehydrogenase and K+ remains unreleased. The particles containing these substances sediment to the same density as synaptosomes. Synaptosomes retaining their internal organelles after hypoosmotic treatment can be seen in electron micrographs. Resealing of disrupted synaptosomes was measured by the inclusion of [14C]sucrose. The resealing is spontaneous, essentially complete (80-90%) within 20 min and not noticeably affected by temperature, pH, or the addition of fusogen. The synaptosome preparation after hypoosmotic disruption will therefore contain some undisrupted synaptosomes with some or all of their complement of cytoplasmic constituents, as well as resealed synaptosomes. The retention of the ability of the hypoosmotically treated preparation to convert [14C]choline to [14C]acetylcholine is demonstrated as an example of the disproportionate effect these undisrupted particles have on its properties.

Ultrastructural Characteristics of Platelets Separated from Plasma by ADP-Induced Aggregation

The utilization of blood platelets in experimentation frequently requires their separation from blood and subsequent resuspension in media of known composition. Several methods are available for preparation of isolated platelets (1-3) by differential centrifugation or gel filtration, but most methods are tedious and time consuming. Often platelets obtained by use of such methods are in a state different functionally and ultrastructurally from that of platelets in plasma (4).Recently Mohammad, Reddick, and Mason (5) reported a method in which platelets were separated from plasma by ADP-induced aggregation, washed several times, and then incubated in a carefully selected medium that resulted in deaggregation of platelets.

Some evidence for a pathway of amino acid incorporation, in rat-liver microsomes, which does not require transfer ribonucleic acid

Rat-liver microsomes which exhibit a high activity for the incorporation of [ 14 C]-leucine into their proteins in the absence of added pH5 precipitate (unwashed microsomes), are stimulated when the pH5 preparation is separated from the microsomes by a dialysis membrane. A requirement for the complete pH5 preparation is obtained when the microsomes are washed by resuspension in medium A (washed microsomes), and in contrast to the unwashed preparation, the pH5 dialysable materials do not increase such incorporation. The low activity of the washed microsomes alone can be significantly increased by the addition of S 4 supernate (obtained by precipitating the pH5 preparation) containing negligible quantities of RNA, and further increases can now be obtained by the action of the dialysable factors from the pH5 preparation. Such results suggest that washing the microsomes removes those areas of activity associated with simulation by the pH5 dialysable materials, and which are replaceable by components of the S 4 supernate. The results have been interpreted to indicate that in the unwashed microsomal preparation there exist two pathways for amino acid incorporation, one of which is independent of s-RNA and which can be stimulated by the pH5 dialysate. In support to this conclusion, it has been shown that supplementation with both pH5 precipitate and S 4 supernate, can result in lower incorporations than would be obtained in the presence of either one of these fractions alone.

Some evidence for a pathway of amino acid incorporation, in rat-liver microsomes, which does not require transfer ribonucleic acid

Rat-liver microsomes which exhibit a high activity for the incorporation of [ 14C]-leucine into their proteins in the absence of added pH5 precipitate (unwashed microsomes), are stimulated when the pH5 preparation is separated from the microsomes by a dialysis membrane. A requirement for the complete pH5 preparation is obtained when the microsomes are washed by resuspension in medium A (washed microsomes), and in contrast to the unwashed preparation, the pH5 dialysable materials do not increase such incorporation. The low activity of the washed microsomes alone can be significantly increased by the addition of S 4 supernate (obtained by precipitating the pH5 preparation) containing negligible quantities of RNA, and further increases can now be obtained by the action of the dialysable factors from the pH5 preparation. Such results suggest that washing the microsomes removes those areas of activity associated with simulation by the pH5 dialysable materials, and which are replaceable by components of the S 4 supernate. The results have been interpreted to indicate that in the unwashed microsomal preparation there exist two pathways for amino acid incorporation, one of which is independent of s-RNA and which can be stimulated by the pH5 dialysate. In support to this conclusion, it has been shown that supplementation with both pH5 precipitate and S 4 supernate, can result in lower incorporations than would be obtained in the presence of either one of these fractions alone.