X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)

Background  

Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs).     

Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction

Polymerase chain reaction (PCR) was successfully carried out inside stable and narrowly distributed water-in-oil nanodroplets with a size of 100-300 nm in diameter. The droplets were obtained by the miniemulsion process. Each aqueous droplet serves as a single nanoreactor for the PCR. It was found that the size of the droplets highly depends on the sonication parameters (i.e., time and amplitude) and that these parameters have a great influence on the final concentration of the PCR product. The parameters were chosen that way that conditions for single molecule chemistry were obtained, since the 3D-space is compartimentalized in small nanoreactors in each of which the same reaction takes place in a highly parallel fashion on every single DNA molecule.     

Boron compartmentation in roots of sunflower plants of different boron status: A study using the stable isotopes 10B and 11B adopting two independent approaches

The intracellular compartmentation of boron (B) in roots of sunflower plants precultured with 100 μ M B (high B) or 1 μ M B (low B) was studied using two independent approaches. In the first approach, short-term efflux studies using the stable isotopes ¹¹B and ¹⁰B were carried out. In roots of high B plants, the calculated concentrations of B (nmol g_FW ⁻¹) were 52.6 in the cell wall, 7.5 in the vacuole, 27.1 in the cytosol and 48.0 in the free space. In roots of low B plants, the concentrations of B (nmol g_FW ⁻¹) were 43.4 in the cell wall, 2.8 in the vacuole, 17.9 in the cytosol and almost zero in the free space. Although the B supply differed by a factor 100, the B concentrations in the cytosol and the vacuole of low B plants were 66 and 37% of the respective concentrations in high B plants. This suggests an additional role for B in plant metabolism, besides its function in the cell wall. In the second approach, root B pools (cell sap and water-insoluble residue) were determined for comparison, and found to be in good agreement with the results from the efflux study.     

Rapid, transient, and dose-dependent expression of hsp70 messenger RNA in the rat brain after morphine treatment

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in micro-opioid receptor (MOR1)-expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature-related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6 degrees), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2 degrees C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that increased expression of Hsp70 after morphine application protects brain structures against potentially hazardous effects of opiates.     

Hsp72 is present in plasma from Holstein-Friesian dairy cattle, and the concentration level is repeatable across days and age classes

Although heat shock proteins (Hsps) are primarily considered as being intracellular, this study identified the presence of Hsp72 in plasma from female Holstein-Friesian dairy cattle. Plasma samples were collected from the same animals at different ages and on different days after calving and accordingly divided into 5 age classes. The age classes were calves less than 235 days of age, young heifers between 235 and 305 days of age, older heifers between 305 and 560 days of age, cows early in lactation, and cows later in lactation. For a subsample of animals within each age class, replicate plasma samples were collected from 1 to 7 days apart to test whether the Hsp72 concentration levels are repeatable on this shorter timescale. Hsp72 was observed in plasma samples from animals of all 5 age classes. For animals with blood samples taken a few days apart, the repeatability (within age class) of the Hsp72 concentration was 0.52 +/- 0.06. Age and days from calving significantly affected the Hsp72 concentration level. The highest Hsp72 level was observed in older heifers (305-560 days of age). The repeatability of Hsp72 concentrations across age classes within animal was 0.22 +/- 0.06. High environmental sensitivity and negative genetic associations between production and health traits in this high-producing breed have been documented earlier. Hsp72 is believed to be strictly stress inducible, and the finding of Hsp72 in plasma indicates that even apparently healthy individuals may experience extrinsic or intrinsic stress (or both).     

Spatial and temporal variations in the trematode component community of the mudsnail Hydrobia ventrosa in relation to the occurrence of waterfowl as definitive hosts

The component community of larval trematodes infecting the mudsnail Hydrobia ventrosa (Montagu) was examined in coastal lagoons of the southern Baltic Sea among different host subpopulations in relation to the structure of the waterfowl community. The 10 trematode species observed represent the families Notocotylidae (1), Echinostomatidae (1 or 2), Heterophyidae (2). Monorchidae (1). Microphallidae (3 or 4), Psilostomatidae (1), and Hemiuridae (1). Eight of these species infect waterfowl as adults. The structure of the trematode communities was similar between sampling sites. Seven trematode taxa were commonly found at all sampling sites. Prevalence values of the 6 most abundant taxa, which infect birds as final hosts, were significantly different between neither sampling sites nor across year. Overall trematode prevalence in H. ventrosa fluctuated seasonally. Prevalence usually peaked in summer between July and September or October. Low prevalences were observed in late winter and early spring. In contrast, the seasonal maximum in waterfowl numbers differed between areas because of significant spatial differences in the bird community structure. The species composition of the component trematode community of H. ventrosa in the coastal lagoons of the southern Baltic Sea is more or less independent of the species composition of the waterfowl community. This independence presumably results from the lack of host specificity in most of the observed trematode species. Otherwise, the low host specificity in combination with the enormous waterfowl diversity in the coastal lagoons might explain the stability of the prevalence pattern of the component trematode community.     

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.     

Nucleic acid-independent retrovirus assembly can be driven by dimerization

The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.