Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabies virus through enhanced immune responses

Granulocyte/macrophage colony-stimulatory factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co-injection of GM-CSF DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold increase. Moreover, GM-CSF co-injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2-type against PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced the T cell-mediated immunity biased to the Th1-type, as judged by the significantly higher level of cytokine IL-2 and IFN-gamma production but not IL-4. When challenged with a lethal dose of PrV, the GM-CSF co-injection enhanced the resistance against a PrV infection. This suggests that co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.     

Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Isolation of Pollutant (Pine Needle Ash)-Responding Genes from Tissues of the Seaweed Ulva Pertusa

Genetic responses of the seaweed Ulva pertusa to pine needle ash have been compared using differential display technique. The tissue viability was assessed to evaluate the stress level with triphenyltetrazolium chloride. Total RNA, from tissues treated in seawater containing ash, was reverse transcribed and amplified by PCR with arbitrary primers. The genetic fragments responding to the stress were selectively isolated from agarose gel and sequenced with a DNA auto sequencer. According to sequence analysis, an ash-inducible gene (342 bp) and an ash-suppressed gene (1690 bp) were identified as hypothetical proteins.     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

Calcineurin is expressed and plays a critical role in inflammatory arthritis

Calcineurin is a calcium-activated phosphatase to mediate lymphocyte activation and neuron signaling, but its role in inflammatory arthritis remains largely unknown. In this study, we demonstrate that calcineurin was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The basal expression levels of calcineurin were higher in the cultured synoviocytes of rheumatoid arthritis patients than those of osteoarthritis patients. The calcineurin activity in the synoviocytes was increased by the stimulation with proinflammatory cytokines such as IL-1beta and TNF-alpha. Moreover, rheumatoid arthritis synoviocytes had an enlarged intracellular Ca(2+) store and showed a higher degree of [Ca(2+)](i) release for calcineurin activity than osteoarthritis synoviocytes when stimulated with either TNF-alpha or phorbol myristate acetate. IL-10, an anti-inflammatory cytokine, failed to increase the Ca(2+) and calcineurin activity. The targeted inhibition of calcineurin by the overexpression of calcineurin-binding protein 1, a natural calcineurin antagonist, inhibited the production of IL-6 and matrix metalloproteinase-2 by rheumatoid synoviocytes in a similar manner to the calcineurin inhibitor, cyclosporin A. Moreover, the abundant calcineurin expression was found in the invading pannus in the joints of mice with collagen-induced arthritis. In these mice, calcineurin activity in the cultured synovial and lymph node cells correlated well with the severity of arthritis, but which was suppressed by cyclosporin A treatment. Taken together, our data suggest that the abnormal activation of Ca(2+) and calcineurin in the synoviocytes may contribute to the pathogenesis of chronic arthritis and thus provide a potential target for controlling inflammatory arthritis.