Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus.

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.     

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high‐throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α‐ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high‐throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.     

Overall charge and local charge density of pectin determines the enthalpic and entropic contributions to complexation with β-lactoglobulin

The complex formation between β-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between β-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the β-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on β-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.     

Formation of filopodia in earthworm (Lumbricus terrestris) coelomocytes in response to osmotic stress

Coelomocytes, the immunodefense cells of the earthworm Lumbricus terrestris, are exposed to changing osmotic pressures as the worm's coelomic fluid responds to fluctuating wet-dry conditions of the surrounding soil. Using light and fluorescence microscopy combined with actin and tubulin disrupting drugs, we determined the effects of changing osmotic pressure on coelomocyte morphology. The coelomocytes from L. terrestris respond to an increase in environmental osmotic pressure from isotonic conditions (170 mOsm) to hypertonic conditions (715 mOsm) by changing from a round/petalloid morphology to a filopodial morphology. Cytoskeletal fluorescent staining studies indicate that for filopodia to form, the actin cortical ring, present in most coelomocytes in isotonic conditions, must be disrupted. Breakdown of the actin ring by exposure to a hypertonic environment or actin disrupting drugs allows the formation of actin or tubulin-based filopodia. The filopodia, or podial-like extensions formed by earthworm coelomocytes, may enable the cells to better explore their environment.     

The first sequenced carnivore genome shows complex host-endogenous retrovirus relationships

Host-retrovirus interactions influence the genomic landscape and have contributed substantially to mammalian genome evolution. To gain further insights, we analyzed a female boxer (Canis familiaris) genome for complexity and integration pattern of canine endogenous retroviruses (CfERV). Intriguingly, the first such in-depth analysis of a carnivore species identified 407 CfERV proviruses that represent only 0.15% of the dog genome. In comparison, the same detection criteria identified about six times more HERV proviruses in the human genome that has been estimated to contain a total of 8% retroviral DNA including solitary LTRs. These observed differences in man and dog are likely due to different mechanisms to purge, restrict and protect their genomes against retroviruses. A novel group of gammaretrovirus-like CfERV with high similarity to HERV-Fc1 was found to have potential for active retrotransposition and possibly lateral transmissions between dog and human as a result of close interactions during at least 10.000 years. The CfERV integration landscape showed a non-uniform intra- and inter-chromosomal distribution. Like in other species, different densities of ERVs were observed. Some chromosomal regions were essentially devoid of CfERVs whereas other regions had large numbers of integrations in agreement with distinct selective pressures at different loci. Most CfERVs were integrated in antisense orientation within 100 kb from annotated protein-coding genes. This integration pattern provides evidence for selection against CfERVs in sense orientation relative to chromosomal genes. In conclusion, this ERV analysis of the first carnivorous species supports the notion that different mammals interact distinctively with endogenous retroviruses and suggests that retroviral lateral transmissions between dog and human may have occurred.