Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.     

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.     

Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species

Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.     

Age and biostratigraphic significance of the Punung Rainforest Fauna, East Java, Indonesia, and implications for Pongo and Homo

The Punung Fauna is a key component in the biostratigraphic sequence of Java. It represents the most significant faunal turnover on the island in the last 1.5 million years, when Stegodon and other archaic mammal species characteristic of earlier Faunal stages were replaced by a fully modern fauna that included rainforest-dependent species such as Pongo pygmaeus (orangutan). Here, we report the first numerical ages for the Punung Fauna obtained by luminescence and uranium-series dating of the fossil-bearing deposits and associated flowstones. The Punung Fauna contained in the dated breccia is of early Last Interglacial age (between 128+/-15 and 118+/-3 ka). This result has implications for the age of the preceding Ngandong Fauna, including Homo erectus remains found in the Ngandong Terrace, and for the timing of Homo sapiens arrival in Southeast Asia, in view of claims for a modern human tooth associated with the Punung breccia.     

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

The phenylanine-glycine (FG)-rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin alpha/beta-mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events.     

Simultaneous purification and polymerization method for bovine serum albumin preparation

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.     

A convenient and efficient protocol for oxidative aromatization of Hantzsch 1,4-dihydropyridines using benzyltriphenylphosphonium peroxymonosulfate under almost neutral reaction conditions

Oxidative aromatization of 4-alkyl or aryl and heterocyclic-substituted derivatives of Hantzsch 1,4-dihydropyridines to the corresponding pyridine derivatives has been studied using benzyltriphenylphosphonium peroxymonosulfate as an oxidant in the presence of BiCl(3) under nearly neutral reaction conditions at ambient temperature.     

Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells

Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the nonexpressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA-mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA-mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA-mediated nuclear localization.     

Recognition of functional genetic polymorphism using ESE motif definition: a conservative evolutionary approach to CYP2D6/CYP2C19 gene variants

Genetica - Although predicting the effects of variants near intron-exon boundaries is relatively straightforward, predicting the functional Exon Splicing Enhancers (ESEs) and the possible effects...     

2q35-rs13387042 variant and the risk of breast cancer: a case–control study

Molecular Biology Reports - Breast Cancer is the most frequent neoplasm diagnosed among women worldwide. Genetic background and lifestyle/environment play a significant role in the disease...