Relative role of low- and high pressure reflexes on sympathetic activity in humans during simulated gravitational stress

In order to determine the relative role of low- and high-pressure reflexes, respectively, on forearm sympathetic nerve activity (fSNA), 10 normal male subjects underwent a 4-step (5 min each) graded lower body negative pressure (LBNP) from -10 to -50 mmHg. Central venous pressure (CVP) and stroke volume gradually decreased (p<0.05), and arterial pulse pressure (PP) abruptly decreased at LBNP of -50 mmHg. Mean arterial pressure (MAP) remained unchanged. Forearm venous plasma norepinephrine concentration (fvNE) increased significantly at LBNP of -35 mmHg (p<0.05) and with a further sharp increase during LBNP of -50 mmHg (p<0.05). High degrees of intra-individual correlations were observed between changes in Log [fvNE] and CVP (r-values from -0.78 to -0.96, p<0.01). We conclude that low-pressure reflexes are the major determinants of fSNA during non-hypotensive gravitational stress (MAP and PP unchanged). When the gravitational stress is more pronounced, a decrease in PP further augments fSNA through inhibition of high-pressure arterial baroreflexes.     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Genome sequence analyses of two isolates from the recent <Emphasis Type="Italic">Escherichia coli</Emphasis> outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EAHEC)

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).     

Supernumerary marker chromosome 5 diagnosed by M-FISH in a child with congenital heart defect and unusual face

We describe a female patient with a small supernumerary marker chromosome (sSMC) present in mosaic and characterized in detail by fluorescence in situ hybridization (FISH) using all 24 human whole chromosome painting probes, multicolor banding (MCB) and subcentromere specific multicolor FISH (subcenM-FISH). The sSMC was demonstrated to be derived from chromosome 5 and the karyotype of our patient was as follows: 47,XX,+mar.ish r(5)(::p13.2 approximately p13.3-->q11.2::) [60%]/46,XX [40%]. Partial trisomy for the proximal 5p and q chromosomal regions is a rare event. A critical region exists at 5p13 for the phenotype associated with duplication 5p. As far as we know, eight similar cases have been published up to now. We describe a new case which, to our knowledge, is the first characterized in such detail. The role of uniparental disomy (UPD) in cases of SMC is also discussed.     

Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT)

Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.     

Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture

Summary Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits with the tissue of origin. Microvasculature was localized in situ by immunohistochemitry in breast samples. From this tissue, collagen-rich stroma and adipose tissue were dissected mechanically and further disaggregated to release microvessel organoids BRENCs were cultured from these organoids in endothelial specific medium and characterized by staining for endothelial markers. Microvessels were a prominent feature of intralobular tissue as evidenced by immunostaining against endothelial specific markers such as CD31, VE-cadherin, and von Willebrand factor (VWF). Double staining against VE-cadherin and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) showed that blood and lymphatic vessels could be distinguished. An antibody against CD31 was used to refine protocols for isolation of microvasculature from reduction mammoplasties. BRENCs retained critical traits even at high passage, including uptake of low-density lipoprotein, and had E-selectin induced upon treatment with tumor necrosis factor-α. The first signs of senescence in passage 14 were accompained by gain of trisomy 11. At passage 18 cells showed chromosomal aberrations and growth arrest as revealed by β-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis.     

Development of pig embryos by nuclear transfer of cultured fibroblast cells

Pig fibroblast cells were transferred to enucleated oocytes by micromanipulation and electrofusion. The donor cells used for nuclear transfer were synchronized in presumptive G0 by serum starvation. In the first experiment, nuclear transfer was performed with fibroblasts that had either a smooth or a rough surface. A significant difference (p < 0.05) in the percentage of chromosome condensation (39.5%, 15/38 and 16.6%, 5/30) and nuclear formation (36.8%, 14/38 and 16.3%, 8/49) was found between the reconstructed embryos derived from the cells with smooth surface and with rough surfaces, respectively. The percentage of chromosome condensation (42.5%, 17/40 and 19.6%, 11/56) and nuclear formation (38.3%, 23/60 and 18.8%, 9/48) were higher (p < 0.05) in reconstructed embryos derived from small (15 microm) donor cells compared to large donor cells (20 microm), respectively. The percentage nuclei at 3 different time points (3, 6, and 9 hours in culture medium) was higher (p = 0.003) in the reconstructed embryos activated by thimerosal and dithiothreitol (20%, 36%, and 41.3%) compared to those without activation treatment (0%, 11.8%, and 22.2%). In addition, there was an increased percentage with nuclei as the time in culture increased from 3 to 9 hours (p = 0.029). The percentages of chromosome condensation (34.6%; 9/26) and nuclear formation (33.3%; 9/27) in nuclear transfer embryos were similar. The rate of blastocysts/morulae development (14.0%; 6/43) was low. However, 2 cavitated embryos (presumptive blastocysts) with 14 and 11 nuclei and 1 morula with 8 nuclei were obtained. This together with the above evidence indicate that the nuclei from pig fibroblast cells can be partially reprogrammed, which suggests that transfer of nuclei from fibroblast cells to in vitro matured oocytes resulting in production of identical or genetically altered pigs may be possible.