Production of infectious human cytomegalovirus virions is inhibited by drugs that disrupt calcium homeostasis in the endoplasmic reticulum

We previously reported that human cytomegalovirus (HCMV) infection induces endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR). Although some normal consequences of UPR activation (e.g., translation attenuation) are detrimental to viral infection, we have previously shown that HCMV infection adapts the UPR to benefit the viral infection (14). For example, UPR-induced translation attenuation is inhibited by viral infection, while potentially beneficial aspects of the UPR are maintained. In the present work, we tested the ability of HCMV to overcome a robust induction of the UPR by the drugs thapsigargin and clotrimazole (CLT), which disrupt ER calcium homeostasis. A 24-h treatment with these drugs beginning at 48, 72, or 96 h postinfection (hpi) completely inhibited further production of infectious virions. HCMV could not overcome the inhibition of global translation by CLT; however, between 48 and 72 hpi, HCMV overcame translational inhibition caused by thapsigargin. Despite the restoration of translation in thapsigargin, the accumulation of immediate-early and early gene products was modestly retarded (50% or less), whereas the accumulation of an early-late and late gene product was significantly retarded. Electron microscopic analysis shows that the drugs severely disrupt the maturation of HCMV virions. This can be accounted for by both the retarded accumulation of late gene products and the drug-induced depletion of ER calcium, which disrupts critical cellular functions needed for maturation.     

Hypertriglyceridemia in lecithin-cholesterol acyltransferase-deficient mice is associated with hepatic overproduction of triglycerides, increased lipogenesis, and improved glucose tolerance

Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr-/- x lcat-/- (double knockout (dko)) mice using the ldlr-/- x lcat+/+ (knock-out (ko)) littermates as control. Mean fasting triglyceride (TG) levels in the dko mice were elevated 1.75-fold compared with their controls (p < 0.002). Both the very low density lipoprotein and the low density lipoprotein/intermediate density lipoprotein fractions separated by fast protein liquid chromatography were TG-enriched in the dko mice. In vitro lipolysis assay revealed that the dko mouse very low density lipoprotein (d < 1.019 g/ml) fraction separated by ultracentrifugation was a more efficient substrate for lipolysis by exogenous bovine lipoprotein lipase. Post-heparin lipoprotein lipase activity was reduced by 61% in the dko mice. Hepatic TG production rate, determined after intravenous Triton WR1339 injection, was increased 8-fold in the dko mice. Hepatic mRNA levels of sterol regulatory element binding protein-1 (srebp-1) and its target genes acetyl-CoA carboxylase-1 (acc-1), fatty acid synthase (fas), and stearoyl-CoA desaturase-1 (scd-1) were significantly elevated in the dko mice compared with the ko control. The hepatic mRNA levels of LXRalpha (lxralpha) and its target genes including angiopoietin-like protein 3 (angptl-3) in the dko mice were unchanged. Fasting glucose and insulin levels were reduced by 31 and 42%, respectively in the dko mice, in conjunction with a 49% reduction in hepatic pepck-1 mRNA (p = 0.014). Both the HTG and the improved fasting glucose phenotype seen in the dko mice are at least in part attributable to an up-regulation of the hepatic srebp-1c gene.     

Quantification of endothelin receptor subtypes in peripheral tissues reveals downregulation of ET(A) receptors in ET(B)-deficient mice

We have previously shown that in homozygous endothelin (ET)(B)-/- deficient mice, ET(A) receptor density is significantly downregulated in the brain by 45%. In these mice, plasma ET-1 levels are elevated. Our aim was to use quantitative autoradiography to establish the distribution of ET receptor subtypes in peripheral tissues from wild-type mice and to measure the density of the ET(A) subtype in ET(B)-/- knockout animals. Our second aim was to test whether deletion of ET(B) receptors, which is associated with elevated plasma levels of ET-1, would also reduce ET(A) expression in the periphery. In longitudinal sections from wild-type mice, the highest densities of ET(A) receptors localized to major organs including the ventricle of the heart, lung, and liver parenchyma. High densities of ET(A) receptors were detected in the smooth muscle layer of the vasculature such as intrarenal vessels as well as the smooth muscle layer and epithelial cells of the gastrointestinal tract. In these tissues, the ET(A) subtype was more abundant, representing between 60% and 100% of the ET receptors. ET(B) receptors predominated in the medulla of kidney, with high densities also localizing to glomeruli within the cortex and to the sinusoids from the liver. Lower densities of ET(B) receptors were also present in the lung, heart, liver, and the smooth muscle layer of the gastrointestinal tract. In ET(B)-/- knockout mice, ET(B) receptors were not detected as expected by either ligand binding or immunocytochemistry. The pattern of ET(A) receptor distribution in the ET(B)-/- knockout mice was similar to the controls, but the density of ET(A) receptors was significantly reduced in the lung by 39%. Diminished responses to the endogenous agonist after repeated stimulation are an important feature of G-protein signaling, preventing potential damage to the overstimulated cell, and it is likely that downregulation occurs in response to higher circulating levels of ET-1.     

Computed tomography analysis of radiostereometric data to determine flexion axes after total joint replacement: Application to the elbow joint

Kinematic analysis for in vivo assessment of elbow endoprostheses requires knowledge of the exact positions of motion axes relative to bony landmarks or the prosthesis. A prosthesis-based reference system is required for comparison between individuals and studies. The primary aim of this study was to further develop an earlier described algorithm for fusion of radiostereometric analysis (RSA) data and data obtained in 3D computed tomography (CT) for application to the elbow after total joint replacement. The secondary aim was to propose a method for marking of prostheses in 3D CT, enabling definition of a prosthesis-based reference system. Six patients with elbow endoprostheses were investigated.The fusion of data made it possible to visualize the motion axes in relation to the prostheses in the 3D CT volume. The differences between two repeated positioning repetitions of the longitudinal prosthesis axis were less than 0.6° in the frontal and sagittal planes. Corresponding values for the transverse axis were less than 0.6° in the frontal and less than 1.4° (in four out of six less than 0.6°) in the horizontal plane.This study shows that by fusion of CT and RSA data it is possible to determine the accurate position of the flexion axes of the elbow joint after total joint replacement in vivo. The proposed method for implant marking and registration of reference axes enables comparison of prosthesis function between patients and studies.     

Branch surface area and its vertical distribution in coastal Douglas-fir

Wood area index (WAI; total surface area of branches and bole per unit of land area) is an important yet often neglected forest structural attribute. Branchwood surface area, in particular, has significant implications for many ecophysiological processes including total respiration and interception of radiation and rainfall. Branch surface area was estimated at the branch-, tree-, and stand-level for 33 Douglas-fir (Pseduotsuga menziesii [Mirb.] Franco) plantations in the Oregon Coast Range. Patterns in WAI, leaf area index (LAI; total surface area of needles per unit of land area), tree area index (TAI=WAI+LAI) and various ratios of these dimensions were then investigated. The main axes of primary branches (those attached to the main stem) comprised 82±13% of total branchwood surface area. Tree surface area (needles + woody tissue) increased with increasing tree size and crown length, and decreased with greater intensity of Swiss needle cast (SNC). At the stand-level, woody surface area increased with greater stand density and decreased with more severe SNC, but on average it constituted 29±12% of total tree surface area. Branchwood surface area and bole surface area contributed equally to WAI. The variation in WAI for a given LAI has important implications for radiation and rainfall attenuation in these stands and for accurate partitioning of intercepted radiation between photosynthetic and non-photosynthetic tissues.     

Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.     

Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodeling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. To study the direct consequences of VSMC apoptosis, we generated transgenic mice expressing the human diphtheria toxin receptor (hDTR, encoded by HBEGF) from a minimal Tagln (also known as SM22alpha) promoter. Despite apoptosis inducing loss of 50-70% of VSMCs, normal arteries showed no inflammation, reactive proliferation, thrombosis, remodeling or aneurysm formation. In contrast, VSMC apoptosis in atherosclerotic plaques of SM22alpha-hDTR Apoe-/- mice induced marked thinning of fibrous cap, loss of collagen and matrix, accumulation of cell debris and intense intimal inflammation. We conclude that VSMC apoptosis is 'silent' in normal arteries, which have a large capacity to withstand cell loss. In contrast, VSMC apoptosis alone is sufficient to induce features of plaque vulnerability in atherosclerosis. SM22alpha-hDTR Apoe-/- mice may represent an important new model to test agents proposed to stabilize atherosclerotic plaques.     

Embryonic development in ballan wrasse Labrus bergylta

Eight primary embryonic developmental stages were assigned to eggs of ballan wrasse Labrus bergylta using key morphological features following standardized nomenclature: Ia, Ib, II, III, IV, V, VI and VI+, reared from single family clutches under comparable environmental conditions in Ireland and Norway. Development in L. bergylta is typical of demersal marine finfish species with a short egg stage. Hatching occurred c. 123 h post-fertilization (hpf) equivalent to 62·5 degree days at 12·2 ± 1·10° C (mean ±s.d.), after which the larvae swam intermittently near the surface of the water column.     

WNT-3a modulates platelet function by regulating small GTPase activity

Here we provide evidence that WNT-3a modulates platelet function by regulating the activity of four key GTPase proteins: Rap1, Cdc42, Rac1 and RhoA. We observe WNT-3a to differentially regulate small GTPase activity in platelets, promoting the GDP-bound form of Rap1b to inhibit integrin-α(IIb)β(3) adhesion, while concomitantly increasing Cdc42 and Rac1-GTP levels thereby disrupting normal platelet spreading. We demonstrate that Daam-1 interacts with Dishevelled upon platelet activation, which correlates with increased RhoA-GTP levels. Upon pre-treatment with WNT-3a, this complex disassociates, concurrent with a reduction in RhoA-GTP. Together these data implicate WNT-3a as a novel upstream regulator of small GTPase activity in platelets.