Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

Background  

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.     

Acute modulation of Ca2+ influx on rat heart by 17beta-estradiol

Estrogens initiate their action by binding to specific intracellular receptors and then acting on gene expression. In addition, there is growing evidence of a direct membrane effect via interaction with a cell surphase receptor. The aim of the present study was to investigate the acute effects of 17beta-estradiol on Ca2+ fluxes through second messenger pathways in rat cardiac muscle. Exposure of rat ventricle to low levels of 17beta-estradiol (10(-12)-10(-8) M) increased 45Ca2+ influx within 1 min (+38%); the response was biphasic, peaking at 2 and 5 min (+60 and +55%, respectively). The effect of the hormone on rat heart seems to be specific since 17alpha-estradiol, dihydrotestosterone, and progesterone were devoid of activity. The effect of 17beta-estradiol (5 min, 10(-10) M) was suppressed by nitrendipine (1 microM) and LaCl3 (10 microM), involving the activation of voltage-dependent Ca2+ channels in the acute increase of rat heart calcium influx by the hormone. 17Beta-estradiol rapidly increased cAMP content and PKA activity of rat cardiac muscle in parallel to the changes in Ca2+ uptake. In addition the cAMP antagonist Rp-cAMPS suppressed 17beta-estradiol-dependent Ca2+ influx. Altogether, the data suggest the involvement of the cAMP/PKA messenger system in the nongenomic modulation of Ca2+ influx in rat cardiac muscle by physiological levels of 17beta-estradiol.     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains

Background

Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers.

Results

The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping.We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population.

Conclusion

The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under “GNU GPL v3”. The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0475-6) contains supplementary material, which is available to authorized users.     

Novel action of estrone on vascular tissue: regulation of NOS and COX activity

The hypothesis tested in the present work is that estrone non-genomically regulates aortic nitric oxide synthase (NOS) and cyclooxygenase (COX) activities in female rats, and that such regulation depends on ovarian function. We found that physiological concentrations of estrone (E(1)) (0.1-10nM) significantly increased nitric oxide (NO) production (133 and 163% above control). The stimulatory action of E(1) on NOS activity was independent of calcium influx since the increase in NO elicited by the hormone was not affected by EGTA or verapamil. When COX activity was measured, we observed that estrone enhanced thromboxane (TXB(2)) production and prostacyclin (PGI(2)) release, but not prostaglandin (PGF(2), PGD(2), and PGE(2)) synthesis. Finally we demonstrated that the hormonal effect on NOS activity was not detected in rat aortic strips (RAS) isolated from animals deprived of ovarian activity (FR(-)) or ovariectomized rats (OVX). These results suggest that estrone exerts a direct, non-genomic action on rat aortic metabolism, which involves NOS and COX activation and depends on ovarian activity.     

Cellular actions of testosterone in vascular cells: Mechanism independent of aromatization to estradiol

In this work we investigated the role of testosterone on cellular processes involved in vascular disease, and whether these effects depend on its local conversion to estradiol. Cultures of rat aortic endothelial and smooth muscle cells in vitro treated with physiological concentrations of testosterone were employed. Testosterone rapidly increased endothelial nitric oxide production. To evaluate whether this non genomic action was dependent on testosterone aromatization we used an aromatase inhibitor. Anastrozole compound did not modify the fast increase in nitric oxide production elicited by testosterone. The hormonal effect was completely blocked by an androgen receptor antagonist (flutamide); meanwhile it wasn′t modified by the presence of an estrogen receptor antagonist (ICI182780).The possibility of intracellular estradiol synthesis was ruled out when no differences were found in estradiol measurements performed in culture incubation medium from control and testosterone treated cells. The 5α-reductase inhibitor finasteride partially suppressed the enhancement in nitric oxide production, suggesting that the effect of testosterone was partially due to dihydrotestosterone conversion. Testosterone stimulated muscle cell proliferation independent of local conversion to estradiol. When cellular events that play key roles in vascular disease development were analyzed, testosterone prevented monocyte adhesion to endothelial cells induced by a proinflammatory stimulus (bacterial lipopolysaccharides), and prompted muscle cell migration in presence of a cell motility inducer. In summary, testosterone modulates vascular behavior through its direct action on vascular cells independent of aromatization to estradiol. The cellular actions exhibited by the steroid varied whether cells were under basal or inflammatory conditions.     

An AFLP-based genetic linkage map of Plasmodium chabaudi chabaudi

Background

Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.

Methods

The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers (used as chromosomal anchors) were organized into linkage groups using Map Manager software.

Results

614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, Plasmodium falciparum

Conclusion

The P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.     

Progesterone and 17 beta-estradiol acutely stimulate nitric oxide synthase activity in rat aorta and inhibit platelet aggregation

The rapid non-genomic stimulatory action of progesterone (Pg) and estradiol (E2) on nitric oxide synthase (NOS) activity of endothelium intact aortic rings and its effect on platelet aggregation was investigated. First we measured the effect of the hormones on platelet aggregation when added to rat aortic strips (RAS) incubated in a PRP. RAS induced an antiaggregatory activity, which was enhanced by the presence of the hormones. The inhibitory action induced by the hormones was evoked in a dose dependent manner (10 pM-100 nM). These effects are specific for progesterone and 17-beta-estradiol, since either testosterone and 17-alpha-estradiol were devoid of activity. The hormones induced rapid responses, producing significant inhibition within 1 to 5 minutes of hormonal exposure. The addition of 10(-5) M L-NAME suppressed the antiaggregatory effect of 1 nM E2 or 10 nM Pg. Furthermore, we specifically quantified the NO generation by the 3H-citrulline technique. 10(-8) M E2 induced 2-fold increase of RAS citrulline production, while the increment induced by 10(-7) M Pg was 55% over control. Preincubation with 10(-5) M L-NAME completely suppressed the stimulatory action of 10(-9) M E2 or 10(-8) M Pg, confirming that the antiaggregatory factor released from the aortic tissue was NO. Preincubation with cycloheximide did not block the increment in NO induced by the hormones. In conclusion the present study provides for the first time evidence of acute, non-genomic effects of Pg on rat aorta NOS activity and platelet aggregation in coincidence with the results obtained with estradiol treatment.     

Stress protein of cyanobacteria CP36: interaction with photoactive complexes and formation of supramolecular structures

Cyanobacterium Anacystis nidulans R2, Synechocystis sp. PCC 6803 (wild-type strain and mutants Delta2 and Delta3 lacking PSII and PSI, respectively), and Synechocystis sp. BO 9201 synthesize the pigment--protein complex CP36 (CPIV-4, CP43') under iron deficiency in the medium. Accumulation of CP36 is accompanied by structural reorganizations in the photosynthetic membranes. Integrating mean times of excitation relaxation (quenching) are 2.2 nsec (CP36), 1 nsec (PSI), and 420 psec (PSII in Fm state). The energy migration between CP36 and the photosystems can be described by a model of a one-layer ring of CP36 around core-complexes. The excitation from CP36 to PSI is transferred within <10 psec. The energy transfer from CP36 to PSII occurs during 170 psec. Cells with low content of CP36 probably contain only a latent fraction of unbound to phycobilisomes PSII which is the analog of PSIIbeta of higher plants. In PSI there are four binding sites for CP36 monomers per RC. PSII can bind up to 32 molecules of CP36 per RC. Cells with a large amount of CP36 contain monomer form of PSII core-complex which can bind eight tetramers of CP36 (8 binding sites). In conditions of iron deficiency only one monomer of a dimer PSII core-complex is destroyed and released chlorophyll is accumulated in CP36. Accumulation of CP36 in A. nidulans cells can be accompanied by membrane stacking which is similar to the stacking in chlorophyll b-containing organisms. The stacking can occur in the region of localization of PSII latent fraction bound to CP36. The membrane stacking shields PSII stromal surfaces from the aqueous phase for activation of electron transfer on the acceptor side of PSII.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.