A faster reconstitution of hematopoiesis after autologous transplantation of hematopoietic cells cryopreserved in 7.5% dimethyl sulfoxide if compared to 10% dimethyl sulfoxide containing medium

Our previous in vitro studies proved a higher clonogenic potential of peripheral blood progenitor cells cryopreserved in 7.5% dimethyl sulfoxide (Me₂SO) than in 10% Me₂SO containing medium. Based on this findings 7.5% Me₂SO cryopreservation medium was introduced to our protocol and both the hematopoietic recovery and infusion-related toxicity were compared with that obtained with standard 10% Me₂SO containing solution. Two cohorts of consecutive patients treated with autologous hematopoietic stem cell transplantation were included in the analysis: 56 patients with PBPCs cryopreserved in 7.5% Me₂SO solution and 52 patients who obtained cells cryopreserved in 10% Me₂SO. Both study groups did not differ significantly with regard to age, diagnosis, and the number of transplanted CD34⁺ cells. The time to leukocyte recovery was shorter for patients in the 7.5% Me₂SO treated group than in the 10% one. Reconstitution of platelets and the frequency of adverse events did not differ in both groups. Reduction of Me₂SO concentration from 10% to 7.5% in cryoprotective mixture has a beneficial impact on leukocyte recovery. These findings require verification in a prospective, randomized trial.     

Preferential interaction of dimethyl sulfoxide and phosphatidyl choline membranes

The interaction free energy of dimethyl sulfoxide (DMSO) and two types phospholipid membranes has been assessed from measurements of vapor pressure. The lipids were phosphatidyl cholines with respectively (14:0/14:0) (DMPC) and (16:0/18:1) (POPC) fatty acid chains. The results were expressed in terms of the iso-osmolal preferential interaction parameter, Γ_μ1, which remained negative under all experimental conditions investigated here. This shows that water-membrane interactions are more favorable than DMSO-membrane interactions. This condition is known as preferential exclusion of DMSO (or preferential hydration of the membrane), and implies that the local (interfacial) concentration of the solute is reduced compared to the bulk. At room temperature and 1 m DMSO, Γ_μ1 was −0.3 to −0.4 for both lipids. This corresponds to a sizable reduction in the DMSO concentration in a zone including at least the first two hydration layers of the membrane. Possible origins of the preferential exclusion are discussed.As a direct consequence of the pronounced preferential exclusion, DMSO generates an osmotic stress at the membrane interface. This tends to stabilize lipid phases of low surface areas and to withdraw water from multilamellar stacks of membranes. Based on this, we suggest that the preferential exclusion of DMSO explains both the modulation of phase behavior and the constriction of multilamellar aggregates induced by this solute.     

Cryopreservation of goldfish fins and optimization for field scale cryobanking

When gametes and embryos are not available, cryobanking of somatic tissues is one possibility to keep a genetic record of fish valuables in a context of biodiversity conservation and animal breeding management. Cryopreservation of whole fin pieces would be more advantageous than the commonly used cryopreservation of cells after fin culture, as it would allow extensive sampling without immediate need for laboratory facilities. The objective of this work was to assess the cryopreservation ability of fin pieces from goldfish (Carassius auratus) and to test whether a laboratory procedure could be adapted to field conditions. Caudal fin explants were cryopreserved in culture medium with 125 mM sucrose and 10% Me₂SO. After 14 days of culture, the frozen–thawed explants showed the same cell growth rate and grew the same somatic cell number as the fresh ones. Cells proliferated inside and around the explants as shown by BrdU labeling. Neither the size of the fin pieces nor the freezer type, −70 °C upright or −20 °C chest, influenced the outcome of cryopreservation. Fin pieces were stored 4 days at 4 °C in dry conditions prior to cryopreservation without alteration of the fin explant culture success. This study demonstrated that field collecting of goldfish fin pieces is possible as whole fin pieces can be stored in standard fridge or be shipped at subzero temperature before they are frozen into a plain −20 °C chest freezer. After incorporation in cryobanks in liquid nitrogen, thawed fin pieces reliably produce somatic cells in cell culture conditions.     

Comparative analysis of transcriptional responses to the cryoprotectants, dimethyl sulfoxide and trehalose, which confer tolerance to freeze–thaw stress in Saccharomyces cerevisiae

We have used microarray analysis to monitor the gene expression profile of Saccharomyces cerevisiae BY4743 in the presence of the cryoprotectants, dimethyl sulfoxide (Me₂SO) and trehalose. Analysis of these profiles suggests that both cryoprotectants increased the expression of genes involved in protein synthesis, ribosomal biogenesis, fatty acid biosynthesis, ergosterol biosynthesis, cell wall biosynthesis, and cellular accumulation of low molecular compounds such as glycerol, arginine and proline. Cryoprotectant treatment reduced the expression of genes involved in the β-oxidation of fatty acids. In addition, Me₂SO increased the expression of genes involved in protein refolding and trehalose increased the expression of genes involved in spore formation. This study supported that exposure to cryoprotectants prior to freezing not only reduce the freeze–thaw damage but also provide various process to the recovery from freeze–thaw damage.     

Conformational features of cepacian: the exopolysaccharide produced by clinical strains of Burkholderia cepacia

Conformational energy calculations and molecular dynamics investigations, both in water and in dimethyl sulfoxide, were carried out on the exopolysaccharide cepacian produced by the majority of the clinical strains of Burkholderia cepacia, an opportunistic pathogen causing serious lung infection in patients affected by cystic fibrosis, The investigation was aimed at defining the structural and conformational features, which might be relevant for clarification of the structure-function relationships of the polymer. The molecular dynamics calculations were carried out by Ramachandran-type energy plots of the disaccharides that constitute the polymer repeating unit. The dynamics of an oligomer composed of three repeating units were investigated in water and in Me2SO, a non-aggregating solvent. Analysis of the time persistence of hydrogen bonds showed the presence of a large number of favourable interactions in water, which were less evident in Me2SO. The calculations on the cepacian chain indicated that polymer conformational features in water were affected by the lateral chains, but were also largely dictated by the presence of solvent. Moreover, the large number of intra-chain hydrogen bonds in water disappeared in Me2SO solution, increasing the average dimension of the polymer chains.     

One-step dilution of open-pulled-straw (OPS)-vitrified mouse blastocysts in sucrose-free medium

Embryos vitrified by the open-pulled-straw (OPS) method are only briefly exposed to cryoprotectants and not fully equilibrated with the cryoprotectant. That being the case, conceivably the post-thawing de- and rehydration processes may be omitted. This would render thawing and dilution in a single step and direct transfer to recipients possible without the need for a microscope and other laboratory equipment. Morphologically intact mouse blastocysts from superovulated 5- to 8-week-old virgin female NMRI mice were vitrified according to a protocol [6] slightly modified from the classical OPS-procedure of Vajta et al. [29] consisting of exposure to 10% dimethyl-sulfoxide (Me₂SO) + 10% ethylene glycol (EG) for 1 min, followed by 20% Me₂SO + 20% EG for 20 s before loading into straws that are plunged into liquid nitrogen. In Group 1, 75 blastocysts were exposed to the standard thawing and dilution regimen involving exposure to three solutions of decreasing sucrose content (Control). In Groups 2, 3 and 4, 75 blastocysts each were transferred, in a single step, to medium at 37 °C containing 0.66, 0.33 or 0 M sucrose, respectively. After 48 h of in vitro culture the proportion of hatched blastocysts was determined. In Group 1, this proportion amounted to 82.7%, in Groups 2, 3 and 4 to 76.0%, 73.3% and 78.7%, respectively (P > 0.05). To examine their potential to continue development in vivo, OPS-vitrified blastocysts thawed according to the regimens of Groups 1 and 4 were transferred to recipients (10 embryos/recipient). In Group 1, 9/10 recipients got pregnant with 4.7 ± 0.6 (mean ± SEM) fetuses, in Group 4, 8/10 recipients with 5.0 ± 0.5 fetuses. The overall embryo survival rate per group was 42% for Group 1 and 40% for Group 4. All fetuses were normally developed and viable and there were no significant differences between groups (P > 0.05). It may be concluded that warming and transfer of OPS-vitrified mouse embryos in a single step in medium devoid of sucrose is feasible, which is tantamount to a substantial simplification of embryo transfer operations.     

Varying amounts of different aldehydes present in the cryoprotectants dimethyl sulphoxide and 1,2-propanediol

Using high-pressure liquid chromatography two cryoprotectant solvents, dimethyl sulphoxide (four manufacturers) and 1,2-propanediol (one manufacturer) were investigated for aldehyde content. Fractionation of the aldehydes by high pressure liquid chromatography identified up to 11 aldehydes and two ketones in both cryoprotectant solvents in varying concentrations, which differed between manufacturer and container type. Of the 11 aldehydes identified, formaldehyde and acetylaldehyde were consistently in the greatest concentrations. As the low molecular weight aldehydes identified contain reactive polarised carbonyl groups they represent a potential source of intracellular damage when used in oocyte cryopreservation.     

Post-cryopreservation viability of the benthic freshwater diatom Planothidium frequentissimum depends on light levels

Over recent years, several planktonic and benthic freshwater diatom taxa have been established as laboratory model strains. In common with most freshwater diatoms the pennate diatom Planothidium frequentissimum suffers irreversible cell shrinkage on prolonged maintenance by serial transfers, without induction of the sexual cycle. Therefore, alternative strategies are required for the long-term maintenance of this strain. Conventional colligative cryopreservation approaches have previously proven unsuccessful with no regrowth. However, in this study using 5% dimethyl sulfoxide (Me₂SO), controlled cooling at 1 °C min⁻¹, automated ice seeding and cooling to −40 °C with a final plunge into liquid nitrogen, viability levels were enhanced from 0.3 ± 0.4% to 80 ± 3%, by incorporating a 48 h dark-recovery phase after rewarming. Omission, or reduction, of this recovery step resulted in obvious cell damage with photo-bleaching of pigments, indicative of oxidative-stress induced cell damage, with subsequent deterioration of cellular architecture.     

Membrane-active host defense peptides--challenges and perspectives for the development of novel anticancer drugs

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means.Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.     

Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability

Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.     

Differential involvement of ERK1-2 and p38MAPK activation on Swiss 3T3 cell proliferation induced by prostaglandin F2alpha

Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha.     

Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A₂ (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly, endogenous coenzymeA was present in the active site. The structure suggests that, contrary to the other prokaryotic NATs, (BACAN)NAT1 possesses a 14-residue insertion equivalent to the “mammalian insertion”, a structural feature considered unique to mammalian NATs. Moreover, (BACAN)NAT1 structure shows marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial NATs.     

Differential interactions of cerebellin precursor protein (Cbln) subtypes and neurexin variants for synapse formation of cortical neurons

The polyisoprenoid alcohols (dolichols and polyprenols) are found in all living organism, from bacteria to mammals. In animal and yeast cells polyisoprenoids are derived from the cytoplasmic mevalonate (MVA) pathway while in plants two biosynthetic pathways, the MVA and the plastidial methylerythritol phosphate (MEP) pathway provide precursors for polyisoprenoid biosynthesis. The key enzymes of polyisoprenoid synthesis are cis-prenyltransferases (CTPs), responsible for construction of the long hydrocarbon skeleton. CPTs elongate a short all-trans precursor, oligoprenyl diphosphate, by sequential addition of the desired number of isopentenyl diphosphate molecules which results in formation of a stretch of cis units. Several genes encoding CPT have been cloned from bacteria, plants and mammals. Interestingly, in Arabidopsis, the tissue-specific expression of ten putative cis-prenyltransferases was observed. In contrast to polyisoprenoid phosphates serving as cofactors in the biosynthesis of glycoproteins, glucosyl phosphatidyl inositol (GPI) anchor or bacterial peptidoglycan, the biological importance of polyprenols and dolichols still remains a question of debate besides their function of reservoir of substrates for kinase. These extremely hydrophobic superlipids are postulated to be involved in intracellular traffic of proteins and in cellular defense against adverse environmental conditions. Recent publications show a direct link between the dolichol biosynthetic pathway and congenital disorders of glycosylation (CDG). These discoveries highlighting the cellular significance of polyisoprenoids simultaneously establish the background for future pharmacological interventions. Our mini-review summarizes the results of recent studies on polyisoprenoids.     

Effects of light environment on the induction of chlorophyll fluorescence in leaves: a comparative study of Tradescantia species of different ecotypes

In this work, using a PAM-fluorimetry technique, we have compared effects of plant adaptation to the light or dark conditions on the kinetics of chlorophyll a fluorescence yield in Tradecantia leaves of several species (Tradescantia albiflora, Tradescantia fluminensis, Tradescantia navicularis, and Tradescantia sillamontana), which represent plants of different ecotypes. Two fluorescence parameters were used to assess photosynthetic performance in vivo: non-photochemical quenching (NPQ) of chlorophyll fluorescence (q_NPQ) determined by energy losses in the light-harvesting antenna of photosystem 2 (PS2), and PS2 operating efficiency (Φ_PSII). Comparative study of light-induced changes in q_NPQ and Φ_PSII has demonstrated that shade-tolerant Tradecantia species (T. albiflora Kunth, T. fluminensis Vell.) reveal higher capacities for NPQ and demonstrate slower transitions between the ‘light-adapted’ and ‘dark-adapted’ states than succulent species T. navicularis and T. sillamontana, which are typical habitats of semi-deserts. We analyze the photosynthetic performance of Tradescantia species in the context of their adaptabilities to variable environment conditions. The ability of shade-tolerant plants to retain a relatively long-term (∼40-60 min) ‘memory’ for illumination history may be associated with the regulatory mechanisms that provide the flexibility of photosynthetic apparatus in response to fluctuations of light intensity.     

The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice

The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity.A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out (−/−), heterozygous (+/−) and wild-type (+/+). A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties.There was no significant difference in the Young’s modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice.Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated.