Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway

Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.     

Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state

Gap junctions coordinate processes ranging from muscle contraction to ovarian follicle development. Here we show that the gap junction protein Zero population growth (Zpg) is required for germ cell differentiation in the Drosophila ovary. In the absence of Zpg the stem cell daughter destined to differentiate dies. The zpg phenotype is novel, and we used this phenotype to genetically dissect the process of stem cell maintenance and differentiation. Our findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual.     

Amplified intergenic locus polymorphism as a basis for bacterial typing of Listeria spp. and Escherichia coli

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.     

Vascular responses in vivo to 8-epi PGF(2alpha) in normal and hypercholesterolemic pigs

Hypercholesterolemia (HC) is characterized by increased circulating 8-epi-prostaglandin-F(2alpha) (isoprostane), a vasoconstrictor, marker, and mediator of increased oxidative stress, whose vascular effects might be augmented in HC. Anesthetized pigs were studied in vivo with electron beam computed tomography after a 12-wk normal (n = 8) or HC (n = 8) diet. Mean arterial pressure (MAP), single-kidney perfusion, and glomerular filtration rate (GFR) were quantified before and during unilateral intrarenal infusions of U46619 (10 ng x kg(-1) x min(-1)) or isoprostane (1 microg x kg(-1) x min(-1)). Basal renal perfusion and function were similar, and isoprostane infusion elevated its systemic levels similarly in normal and HC (333 +/- 89 vs. 366 +/- 48 pg/ml, respectively, P < 0.01 vs. baseline). Both drugs markedly and comparably decreased cortical perfusion and GFR in both groups, whereas medullary perfusion decreased significantly only in HC. Moreover, MAP increased significantly only in HC (+9 +/- 3 and +11 +/- 3 mmHg, respectively, P相似文献   

Workplace Digital Health Is Associated with Improved Cardiovascular Risk Factors in a Frequency-Dependent Fashion: A Large Prospective Observational Cohort Study

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the US. Emerging employer-sponsored work health programs (WHP) and Digital Health Intervention (DHI) provide monitoring and guidance based on participants’ health risk assessments, but with uncertain success. DHI–mobile technology including online and smartphone interventions–has previously been found to be beneficial in reducing CVD outcomes and risk factors, however its use and efficacy in a large, multisite, primary prevention cohort has not been described to date. We analyzed usage of DHI and change in intermediate markers of CVD over the course of one year in 30,974 participants of a WHP across 81 organizations in 42 states between 2011 and 2014, stratified by participation log-ins categorized as no (n = 14,173), very low (<12/yr, n = 12,260), monthly (n = 3,360), weekly (n = 651), or semi-weekly (at least twice per week). We assessed changes in weight, waist circumference, body mass index (BMI), blood pressure, lipids, and glucose at one year, as a function of participation level. We utilized a Poisson regression model to analyze variables associated with increased participation. Those with the highest level of participation were slightly, but significantly (p<0.0001), older (48.3±11.2 yrs) than non-participants (47.7±12.2 yr) and more likely to be females (63.7% vs 37.3% p<0.0001). Significant improvements in weight loss were demonstrated with every increasing level of DHI usage with the largest being in the semi-weekly group (-3.39±1.06 lbs; p = 0.0013 for difference from weekly). Regression analyses demonstrated that greater participation in the DHI (measured by log-ins) was significantly associated with older age (p<0.001), female sex (p<0.001), and Hispanic ethnicity (p<0.001). The current study demonstrates the success of DHI in a large, community cohort to modestly reduce CVD risk factors in individuals with high participation rate. Furthermore, participants previously underrepresented in WHPs (females and Hispanics) and those with an increased number of CVD risk factors including age and elevated BMI show increased adherence to DHI, supporting the use of this low-cost intervention to improve CVD health.     

Cell encapsulation utilizing PEG-fibrinogen hydrogel supports viability and enhances productivity under stress conditions

Recent advances in the bioengineering field have introduced new opportunities enabling cell encapsulation in three-dimensional (3D) structures using either various natural or synthetic materials. However, such hydrogel scaffolds have not been fully biocompatible for cell cultivation due to the lack of physical stability or bioactivity. Here, we utilized a uniquely fabricated semi-synthetic 3D polyethylene glycol-fibrinogen (PEG-Fb) hydrogel scaffold, which exhibits both high stability and high bioactivity, to encapsulate HEK293 cells for the production of human recombinant acetylcholine esterase (AChE). To examine the beneficial bioactive effect of the PEG-Fb scaffold over 2D surfaces, an experimental system was established to compare the viability, proliferation and AChE secretion of encapsulated cells versus non-encapsulated surface-adherent cells in serum starvation. Our results show that the transfer of surface-adherent HEK293 cells from fully enriched medium with 10% FCS to 0.2% FCS resulted in an eightfold reduction in cell number and a fourfold reduction in AChE production. In contrast, the encapsulated cells were highly viable and about twofold more efficient in AChE production. In addition, they had round morphology with a twofold larger cell diameter, supporting the observation of increased AChE production. These results suggest a role of the PEG-Fb scaffold in providing a supportive microenvironment in reduced serum conditions that enhances encapsulated cell functions, opening new directions to study the implementation of this platform in large-scale pharmaceutical protein production.     

Early changes in coronary artery wall structure detected by microcomputed tomography in experimental hypercholesterolemia

Changes in the structure of the artery wall commence shortly after exposure to cardiovascular risk factors, such as hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful in evaluation of the factors that instigate atherosclerosis and its pathomechanisms. The present study tested the hypothesis that early morphological changes in coronary arteries of hypercholesterolemic (HC) pigs can be detected using the novel X-ray contrast agent OsO(4) and three-dimensional micro-computed tomography (CT). Two groups of pigs were studied after they were fed a normal or an HC (2% cholesterol) diet for 12 wk. Hearts were harvested, coronary arteries were injected with 1% OsO(4) solution, and cardiac samples (6-mum-thick) were scanned by micro-CT. Layers of the epicardial coronary artery wall, early lesions, and perivascular OsO(4) accumulation were determined. Leakage of OsO(4) from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor in corresponding histological cross sections. OsO(4) enhanced the visualization of coronary artery wall layers and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO(4) in HC was correlated with increased vascular endothelial growth factor expression and suggested increased microvascular permeability. The use of OsO(4) as a contrast agent in micro-CT allows three-dimensional visualization of coronary artery wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.     

Impaired myocardial perfusion reserve in experimental hypercholesterolemia is independent of myocardial neovascularization

Our objective was to investigate the functional role of hypercholesterolemia-associated myocardial neovascularization in early atherosclerosis using the antiangiogenic thalidomide. Experimental atherosclerosis is characterized by myocardial neovascularization, associated with a decrease in myocardial perfusion response to challenge, coronary endothelial dysfunction, and high oxidative stress. However, the functional significance of these neovessels is not known. Three groups of pigs (n = 6 each) were studied after 12 wk of normal or hypercholesterolemic diet without (HC) or with thalidomide (HC + Thal). Myocardial perfusion and permeability were assessed at baseline and in response to cardiac challenge, using electron beam computed tomography, and coronary endothelial function was assessed using organ chambers. Myocardial samples were scanned ex vivo with a three-dimensional microscopic computed tomography scanner, and the spatial density of the myocardial microvessels was quantified. Growth factors and oxidative stress were measured in the myocardial tissue. As a results of these procedures, myocardial perfusion response to adenosine and dobutamine was blunted in both HC and HC + Thal pigs compared with normal pigs (P < 0.05, HC and HC + Thal vs. normal) as was the coronary endothelial function. Myocardial permeability response to adenosine was increased in both HC and HC + Thal pigs compared with normal pigs (P < 0.05, HC and HC + Thal vs. normal, and HC + Thal vs. HC). The microvascular density was increased in HC pigs compared with normal pigs but normalized in HC + Thal pigs (P < 0.001 HC vs. normal and HC + Thal). HC + Thal pigs showed decreased expression of Flk-1 and basic FGF but increased expression of VEGF compared with normal and HC pigs. Oxidative stress was increased in both HC and HC + Thal pigs compared with normal pigs. In conclusion, chronic administration of thalidomide attenuates myocardial neovascularization in experimental HC pigs without affecting myocardial perfusion response to stimulation. This suggests that the myocardial neovascularization may not contribute to the attenuated myocardial perfusion response in hypercholesterolemia.