The Transcriptional Corepressor NAB2 Inhibits NGF-induced Differentiation of PC12 Cells

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta 1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21WAF1, a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21WAF1 restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF

Cloning, characterization and regulation of expression of a cold-acclimation-specific gene, cas18, in a freezing tolerant cultivar of alfalfa

Cold-acclimation-specific (CAS) gene expression was examined by screening a cDNA library prepared from poly(A)$sp+$ RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medicago falcata cv Anik). Three distinct CAS cDNA clones, pSM784, pSM2201, and pSM2358 were isolated. The genes corresponding to all three clones are coordinately induced by cold. Expression of these genes is not triggered by other stress treatments such as heat shock, water stress, wounding, or treatment with exogenous ABA. A positive correlation was observed between the level of expression of each gene and the degree of freezing tolerance of four alfalfa cultivars.A full-length cDNA clone for the most abundantly-expressed gene, cas18 was isolated and sequenced. The deduced polypeptide, CAS18, is relatively small (167 amino acids), is highly hydrophillic, rich in glycine and threonine, and contains two distinctive repeat elements. It exhibits homology with members of the LEA/RAB/Dehydrin gene family--proteins which accumulate in response to water stress or abscisic acid (ABA). The cas18 cDNA hybridizes to three transcripts of 1.6, 1.4 and 1.0 kb in cold acclimated seedlings and cell cultures. The clone described here, Acs784, corresponds to the 1.0 kb transcript.Expression of this gene is 30-fold greater in cold-acclimated cells than in nonacclimated cells after one week of low temperature treatment. Return to room temperature (deacclimation) results in the rapid disappearance of the three transcripts within just 5 hours. Studies of nuclear "run-on" transcription and transcript stability show that low temperature regulates the expression of cas18 at both the transcriptional and post-transcriptional levels

Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination

Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection. An immunoproteomics approach was used to survey the repertoire of antibodies in sera from individuals who had contracted tularemia during two outbreaks and individuals from two geographical areas who had been vaccinated with NDBR Lot 11 or Lot 17 LVS. These data showed a large overlap in the antibodies generated in response to tularemia infection or LVS vaccination. A total of seven proteins were observed to be reactive with 60% or more sera from vaccinees and convalescents. A further four proteins were recognised by 30\u201360% of the sera screened. These proteins have the potential to serve as markers of vaccination or candidates for subunit vaccines.Peer reviewed: YesNRC publication: Ye

A U.S. Government-Coordinated Effort to Leverage Non-Human Primate Data to Facilitate Ebolavirus Vaccine Development

A United States Government (USG) interagency group, the Filovirus Animal Non-Clinical Group (FANG), has been established to support the development of biodefense medical countermeasures (MCMs). As both vaccines and therapeutics are licensed using “non-traditional pathways”, such as the U.S. Food and Drug Administration’s (FDA) Animal Rule (AR), non-human primate (NHP) models and associated assays have been developed and standardized across BSL4 testing sites to evaluate candidate products. Vaccine candidates are evaluated using these NHP models, and through this public–private partnership, a meta-analysis of NHP control data has been conducted and submitted to the FDA as a master file. This is an example of how existing NHP control data can be leveraged in lieu of conducting separate natural history studies at multiple testing facilities to demonstrate the consistency of a standardized animal model for vaccine development. As a result, animal use can be minimized and the duplication of effort avoided, thus reducing the amount of time needed to conduct additional studies, as well as the cost of vaccine candidate development. This successful strategy may be applied to other pathogens of high consequence for vaccine development, and shows how strategic preparedness for biodefense can be leveraged in response to outbreaks and public health emergencies

Stimulation indices for the proliferative responses of human PBMC to recall stimulation with ffLVS for five days.

<p>Median values ± SEM per donor group of 12–16 individuals are shown. “pat” indicates convalescent tularemia patients, “vc” LVS vaccinees, and “nv” naïve individuals.</p