Efficient production of nuclear transferred rat embryos by modified methods of reconstruction

In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring.     

Endothelial cells as early sensors of pulmonary interstitial edema.

We studied responses of endothelial and epithelial cells in the thin portion of the air-blood barrier to a rise in interstitial pressure caused by an increase in extravascular water (interstitial edema) obtained in anesthetized rabbits receiving saline infusion (0.5 ml.kg(-1).min(-1) for 3 h). We obtained morphometric analyses of the cells and of their microenvironment (electron microscopy); furthermore, we also studied in lung tissue extracts the biochemical alterations of proteins responsible for signal transduction (PKC, caveolin-1) and cell-cell adhesion (CD31) and of proteins involved in membrane-to-cytoskeleton linkage (alpha-tubulin and beta-tubulin). In endothelial cells, we observed a folding of the plasma membrane with an increase in cell surface area, a doubling of plasmalemma vesicular density, and an increase in cell volume. Minor morphological changes were observed in epithelial cells. Edema did not affect the total plasmalemma amount of PKC, beta-tubulin, and caveolin-1, but alpha-tubulin and CD-31 increased. In edema, the distribution of these proteins changed between the detergent-resistant fraction of the plasma membrane (DRF, lipid microdomains) and the rest of the plasma membrane [high-density fractions (HDFs)]. PKC and tubulin isoforms shifted from the DRF to HDFs in edema, whereas caveolin-1 increased in DRF at the expense of a decrease in phosphorylated caveolin-1. The changes in cellular morphology and in plasma membrane composition suggest an early endothelial response to mechanical stimuli arising at the interstitial level subsequently to a modest (approximately 5%) increase in extravascular water.     

Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis

The aim of this study was to analyze the type of immune response (Th1, Th2) and protein composition of bronchoalveolar lavage (BAL) of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Flow cytometry analysis of intracellular cytokines revealed different patterns: in IPF and SSc Th2 profiles were predominant, whereas in sarcoidosis Th1 prevailed. The proteomic analysis of BAL fluid (BALF) showed that there were quantitative differences between the three diseases. These were more evident between sarcoidosis and IPF, confirming our previous observations, whereas SSc had an intermediate profile between the two, however with some peculiarities. Comparison of BALF protein maps, constructed with the same quantity of total proteins, enabled us to identify the main profiles of the three diseases: an increase in plasma protein prevalent in sarcoidosis and also present in SSc, though for fewer proteins with respect to IPF and a greater abundance of low molecular weight proteins, mainly locally produced, in IPF. These findings are in line with the different pathogenesis of these diseases: IPF is considered a prevalently fibrotic disorder limited to the lung, with intense local production of functionally different proteins, whereas sarcoidosis and SSc are systemic immunoinflammatory diseases.     

Biodegradable polymers from renewable sources: rheological characterization of hemicellulose-based hydrogels

Hemicellulose-based hydrogels were prepared by radical polymerization of 2-hydroxyethyl methacrylate or poly(ethylene glycol) dimethacrylate with oligomeric hydrosoluble hemicellulose modified with well-defined amounts of methacrylic functions. The polymerization reaction was carried out in water at 40 degrees C using a redox initiator system. The hydrogels were in general elastic, soft, and easily swellable in water. Their viscoelastic properties were determined by oscillatory shear measurements on 2 mm thick hydrogels under a slight compression to avoid slip, over the frequency range 10(-1) to 10(2). The rheological characterization indicated that the elastic response of the hydrogels was stronger than the viscous response, leading to the conclusion that the hydrogel systems displayed a predominantly solid-like behavior. The curves showed an increase in shear storage modulus with increasing cross-linking density. The nature of the synthetic comonomer in the hemicellulose-based hydrogels also influenced the shear storage modulus. Comparison of hemicellulose-based hydrogels with pure poly(2-hydroxyethyl methacrylate) hydrogels showed that their behaviors were rather similar, demonstrating that the synthetic procedure made it possible to prepare hemicellulose-based hydrogels with properties similar to those of pure poly(2-hydroxyethyl methacrylate) hydrogels.     

Unconventional method based on circular dichroism to detect peanut DNA in food by means of a PNA probe and a cyanine dye

In this paper we report an innovative and unconventional method based on circular dichroism for the identification of peanut DNA in food, which can be detected after PCR amplification at the nanomolar level by using an achiral PNA probe complementary to a tract of the peanut Ara h 2 gene and an achiral 3,3'-diethylthiadicarbocyanine dye [DiSC(2)(5)]. Peanuts are one of the most common causes of severe allergic reactions to foods and are particularly dangerous when they are "hidden" (undeclared) in food. For better protection of consumers, detection methods are required to specifically detect the presence of hidden allergens in a wide variety of food items. Alternative to the detection of the proteins is the determination of species-specific DNA, which is more resistant to technological treatments. PNAs are very specific probes able to recognize DNA sequences with high affinity and evidence for the binding can be obtained by using the DiSC(2)(5) dye, which aggregates onto the PNA-DNA duplex giving rise to a characteristic visibile band at 540 nm. Because the PNA-DNA duplex is in a right-handed helical conformation, the aggregation of the dye to the duplex gives also rise to a strong CD signal in the 500-600 nm region with a strong exciton coupling due to the formation of multimeric species, since the handedness of the helix is transferred to the dye aggregate. The dye does not interact with the free single-stranded DNA and although aggregating on the achiral PNA, this interaction is obviously not detectable by circular dichroism. Thus, only the formation of the PNA-DNA duplex, which takes place only upon specific Watson-Crick hydrogen binding between the PNA and the DNA bases, is detected, ensuring a very high specificity and sensitivity. The method has been optimized in a model system by using a synthetic oligonucleotide complementary to the PNA probe, showing that the intensity of the signal is linearly related to the amount of the DNA. The optimized method has been applied to the identification and quantitation of DNA extracted and amplified by PCR from peanuts and from peanut-containing foods, allowing for a very sensitive detection at a very low level (few pmol).     

Oceanimonas smirnovii sp. nov., a novel organism isolated from the Black Sea

A slightly creamy, melanogenic, gram-negative, aerobic bacterium was isolated from seawater sample collected in the Karadag Natural Reserve of the Eastern Crimea, the Black Sea. The novel organism was chemoorganotrophic, had no obligate requirement in NaCl, tolerated to 12% NaCl, grew between 10 and 45 degrees C, was slightly alkaliphilic, and was not able to degrade starch, gelatin, agar, and Tween 80. 16S rRNA gene sequence-based analyses of the new organism revealed that Oceanimonas doudoroffii ATCC 27123T, Oceanimonas baumanii ATCC 700832T, and Oceanisphaera litoralis DSM 15406T were the closest relatives (similarity around 97%-96%). The G + C content of the DNA of the strain 31-13T was 55.5mol%. Phosphatidylethanolamine (49.0%), phosphatidylglycerol (41.8%), and diphosphatidylglycerol (9.2%) were the predominant phospholipids. The major fatty acids were 16:0 (24.1%), 16:1omega7 (40.3%), and 18:1omega7 (29.2%). On the basis of the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the bacterium be classified as a novel species; the name Oceanimonas smirnovii sp. nov. is proposed. The type strain is 31-13T (UCM B-11076T = LMG 22147T = ATCC BAA-899T).     

Biochemical and heart adaptations to physical training and supplementation with amino acids

This study evaluated the role of amino acids supplementation on the heart's adaptation under extensive training conditions. Sixty active athletes (bicyclists and swimmers) were separated into 2 groups: 30 were given amino acid mixture (1 g per 10 kg of body weight) for a period of 1 month, and the other 30 were given placebo for the same duration (control group). In the same time period, 20 subjects of similar age not engaged in physical training or sports activities were used as the additional control group. Blood concentrations of alanine transaminase (ALT), asparagine transaminase, lactate dehydrogenase (LDH), gamma-glutamil transpeptidase, alkaline phosphatase (ALP), amylase, triglycerides, albumin, interleukin-6 (IL-6), and interleukin-10 (IL-10) were determined for all subjects before and after the intervention period. Concentrations of LDH and ALP were increased, but concentrations of ALT, albumin, and triglycerides were decreased in the blood of trained athletes compared with healthy subjects not engaged in sports activities. In the athletes, some increases in IL-6 levels were noted; however, they were significantly (p < 0.05) lower than in patients with myocardiodystrophy. The values of IL-10 in athletes were higher than concentrations of IL-10 in patients with myocardiodystrophy but still lower than the normal values. The inhibition of IL-10 in blood may play an important role in the induction of apoptosis in cells of the heart muscle. After amino acid supplementation, the athletes' values for albumin, triglycerides, IL-10, LDH, and ALP were significantly increased compared with the post-placebo control groups. Enzyme activities of other enzymes remained unchanged in all groups. Histological data from a secondary study of actual heart tissue showed that the amino acids supplementation may have inhibiting effects on myocardial apoptosis. The criteria of efficiency of the amino acids supplementation were defined by the albumin, IL-6, and IL-10 concentrations.     

Spatial changes in carbon and nitrogen stable isotopes of the plankton food web in a saline lake ecosystem

We investigated spatial changes in the isotope ratios of the plankton food web in Lake Chany, Siberia, Russia, especially at an estuarine transition zone of the lake. The δ¹³C values of particulate organic matter (POM) varied among the sampling sites, and increased with increasing pH of the lake water. This may reflect a shift by phytoplankton from using CO₂ to using bicarbonate for photosynthesis with increasing pH. The δ¹³C values of zooplankton community also changed at each site along with those of the POM. This was indicative of carbon isotope changes of plankton food webs between the stations along an environmental gradient.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization

Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.