Controllability is an important determinant of the effects of stress on behavior. We trained mice with escapable (ES) and inescapable (IS) shock and examined behavioral freezing and Fos expression in brain regions involved in stress to determine whether stressor controllability produced differential activation of these regions. Mice (C57BL/6J) were trained to escape footshock by moving to a safe chamber in a shuttlebox. This terminated shock for both ES mice (n=5) and yoked-control mice receiving IS (n=5). Handling control (HC) mice (n=5) experienced the shuttlebox, but never received footshock. Training took place on three days (20 trials per day, 0.2 mA, 5.0 sec maximum duration, 1.0 min interstimulus interval). On day 3, the animals were killed two h after training and the brains were processed for Fos expression in the amygdala, hypothalamic paraventricular nucleus (PVN), laterodorsal tegmental nucleus, locus coeruleus and dorsal raphe nucleus. Fos expression after IS was greater than after ES and HC in all regions (p < .05). Fos expression after ES was greater than HC only in PVN (p < .05). Freezing in ES mice was equal to or greater than in IS mice whereas HC mice showed minimal freezing. Differential activation of brain regions implicated in stress may, in part, account for differences in behavior in the aftermath of uncontrollable and controllable stress.
AimsDesigning therapeutics against the HIV envelope glycoprotein (Env) is only as accurate as the structure of the Env they are targeting. Conserving the structure of the Env trimer is crucial for proper experimental assessment of antibody binding and neutralization. However, Env is notably difficult to express by transfection of a recombinant Env plasmid. To increase surface expression, researchers commonly utilize c‐tail mutants of the gp41 transmembrane glycoprotein of HIV‐1, but mutations and deletions in this region can impact the overall conformation and stability of the Env trimer. Multiple studies have shown that while tail mutants have higher Env surface expression, they are easier to neutralize and have altered trimer conformations compared with wild‐type Env found in vivo on infected cells. To assess and characterize native cell surface Env structures, we sought a protocol that could reliably detect wild‐type Env surface expression by flow cytometry.Methods and resultsBy avoiding fetal bovine serum–based buffers, significantly increasing the amounts of transfected plasmid and Env‐specific antibody and by selecting a bright, biotin + streptavidin‐PE detection system, we were able to increase the surface expression of transfected Env protein.ConclusionThis protocol will allow for more precise assessment of antibody binding, epitope exposure, and Env structure, all of which will contribute to designing more effective vaccines and immunotherapeutics.
No abstract
5145 Background: Increased AR level, characteristic of advanced PC, has been linked to AI growth while its down-regulation to restored androgen-dependence (AD). However, the mechanisms of AR overexpression remain in dispute. The objectives were to study the proteins comprising a novel ARS that binds to 5’-UTR of AR gene, which is partially lost in AI LNCaP cells; the status of ARS proteins in AD and AI human specimens and therapeutic modulation of ARS proteins. Methods: Parental AD LNCaP cells and its AI- derivative expressing 4 fold more AR-mRNA and protein than parental AD LNCaP were used to isolate, in vitro/in vivo characterize and therapeutically modulate ARS proteins and cell growth. NYU Tissue Bank provided human samples. Assays: column chromatography, SDS-PAGE, mass spectrometry, EMSA, Southwersten-Western, ChIP, cDNA array, real time PCR, si-RNA, immunohistochemistry. Results: Pur alpha (Pura) and hnRNP-K are part of ARS complex that binds in vitro and in vivo to a defined DNA sequence in 5’UTR of AR gene. AI cells with high AR had 3 times less Pura: its forced expression lowered AR levels. Pura knockdown in AD cells yielded higher AR levels and AI growth. Hormone-naive human PC specimens had significantly increased AR (0.0317) and lower Pura-RNA (0.0317) than hormone resistant PCs. In-vivo binding of Pura (ChIP) to 5’UTR was also reduced (p=0.0028). Histone deactetylase inibitors (HDACI) increased binding of Pura to 5’UTR, decreased AR levels and inhibited AI-growth of LNCaP cells. Conclusions: We show that AR over-expression and AI-growth of a hormone resistant PC cell line are affected by a loss of a repressor complex that binds to 5’-UTR of AR gene. Pura is a crucial part of this complex. We have convincing evidence obtained in hormone naive and resistant human samples to indicate that similar mechanism might be responsible for human PC progression. We determined HDACI can restore Pura levels and androgen-dependence. No significant financial relationships to disclose.
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