Cloned endothelium derived from autoimmune vascular disease retain structural and functional characteristics of normal endothelial cells.

MRL/1pr mice demonstrate anatomic specificity in their development of vasculitis including the small- and medium-sized muscular arteries of the mesentery. To define the functional role of endothelium in vasculitis, we have cloned endothelial cells derived from inflamed small- and medium-sized arteries. Primary cells were derived by enzymatic dispersement and endothelial cells were selected by utilizing a combination of specific culture conditions. Cloned endothelium were developed utilizing limiting dilution cultures supplemented by endothelial cell growth factor. The cloned endothelial cells express many structural features of mature endothelial cells including Factor VIII-RA, non-muscle-specific actin, and Weibel-Palade bodies. Functionally, the clones express functional receptors for the scavenger pathway for LDL metabolism. The cells do not express Class I MHC antigens; however, IFN-beta and IFN-gamma stimulate Class I MHC expression after 24 h, which induces lysis of virus-infected cloned endothelium by Class I-restricted virus-primed T cells. In direct contrast to site-identical vascular smooth muscle cells (VSMCs), endothelial cells do not spontaneously express Class II MHC antigens, nor do they secrete biologically relevant levels of IL-1 unless triggered by lipopolysaccharide. The availability of site-specific cloned endothelium along with cloned VSMCs from autoimmune mice should resolve major experimental controversies involving the pathophysiology of inflammatory vascular disease.     

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

The application of GIS and spatiotemporal analyses to investigations of unusual marine mammal strandings and mortality events

In 2006–2007, an unusually high number of harbor porpoises (Phocoena phocoena) stranded along the Washington and Oregon coastlines. Spatiotemporal analyses were used to examine their ability to detect clusters of porpoise strandings during an unusual mortality event (UME) in the Pacific Northwest using stranding location data. Strandings were evaluated as two separate populations, outer coast and inland waters. The presence of global clustering was evaluated using the Knox spatiotemporal test, and the presence of local clusters was investigated using a spatiotemporal scan statistic (space–time permutation). There was evidence of global clustering, but no local clustering, supporting the hypothesis that strandings were due to more varied etiologies instead of localized causes. Further analyses at subregional levels, and concurrently assessing environmental factors, might reveal additional geographic distribution patterns. This article describes the spatial analytical tools applied in this study and how they can help elucidate the spatiotemporal epidemiology of other UMEs and assist in determining their causes. More than one spatial analytical technique should be used if the study objective is to detect and describe clustering in time and space and to generate hypotheses regarding causation of marine mammal disease and stranding events.     

Effect of solution non-ideality on erythrocyte volume regulation

A non-ideal, hydrated, non-dilute pseudo-binary salt-protein-water solution model of the erythrocyte intracellular solution is presented to describe the osmotic behavior of human erythrocytes. Existing experimental activity data for salts and proteins in aqueous solutions are used to formulate van Laar type expressions for the solvent and solute activity coefficients. Reasonable estimates can therefore be made of the non-ideality of the erythrocyte intracellular solution over a wide range of osmolalities. Solution non-ideality is shown to affect significantly the degree of solute polarization within the erythrocyte intracellular solution during freezing. However, the non-ideality has very little effect upon the amount of water retained within erythrocytes cooled at sub-zero temperatures.     

An experimental comparison of intracellular ice formation and freeze-thaw survival of hela S-3 cells

A small-volume fluorescent dye viability assay has been successfully applied to a conduction cryomicroscope freezing-thawing stage as a means of determining post-thaw survival of the nucleated mammalian cell HeLa S-3. The survival signature for HeLa S-3 cells has been determined, revealing an optimum cooling rate of −30 °C/min where the maximum survival is 30%. No cells survive for cooling rates greater than −128 °C/min and the decreased survival at supraoptimal cooling rates coincides with a linear increase in the percentage of cells containing intracellular ice from 0% at −16 °C/min to 100% at −128 °C/min.Although no data were taken to identify increased salt concentration as the mechanism responsible for cell injury at suboptimal cooling rates, the post-thaw leakage of intracellular fluorescent dye at these rates takes approximately 4–10 min as opposed to instantaneous release of dye for cells which contain ice at the high cooling rates. This indicates two modes of damage.Cell number density has been identified as an important parameter in freezing studies since survival can be enhanced at slow rates by packing cells together in groups. Packing also causes a greater fraction of the cells in a sample to have intracellular ice present, thus decreasing survival at the faster rates. These responses can be explained by assuming that the outer cells in a group protect the inner ones from solution damage at slow rates, yet restrict water flux from the inner cells at faster rates, causing an increased likelihood of intracellular ice formation. Both of these results are consistent with the dual-mechanism freezing damage theory proposed by Mazur.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.