Amino acid residues at the N- and C-termini are essential for the folding of active human butyrylcholinesterase polypeptide

Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200 mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2 x LD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation

Abstract WP213: The Effect Of Nicotine Withdrawal On Stroke Outcome In Female Rats

Byline: Shahil H Patel, Neurology, Univ of Miami Miller Sch of Medicine, Miami, FL; Isabel Saul; Kunjan R Dave; Miguel A Perez-Pinzon; Ami P Raval Significance: Smoking-derived nicotine (N) is known to synergistically magnify the risk and severity of cerebral ischemia in females. Most importantly, smoking is the one preventable risk factor and giving up smoking reduces the risk for cerebral ischemia. However, how long the harmful effects of N on the brain persist after women stop smoking is unknown. In a laboratory study using an animal model of cerebral ischemia, we demonstrated that N alters brain energy metabolism and thus exacerbates ischemic brain damage. Therefore, the current study aims to investigate how long after N withdrawal (NW) N toxicity on brain energy metabolism persists and its impact on stroke outcomes in female rats. Methods: Female Sprague-Dawley rats (n=8/group) were randomly exposed to either saline or N (4.5 mg/kg) for 16-21 days after which point, they were withdrawn from N exposure and able to recover for 0, 15, or 30 days. These rats were then randomly assigned to either have their cortical tissue collected for global metabolomic (Metabolon Inc) and Western blot analysis or undergo a sham surgery or transient middle-cerebral artery occlusion (tMCAO; 90 min). Post-stroke cognition was tested with contextual fear conditioning at month following tMCAO, subsequently the brains were collected for infarct quantification. Results: Analysis of the metabolomics data revealed an increase in carbohydrate metabolites in the 30-day NW group when compared to the N-exposed group, suggesting persistence of N toxicity in the brain. Furthermore, fear conditioning data revealed a significantly lower freezing time in all NW groups when compared to the saline group implying that spatial memory deficits persist even after 30 days of NW. Lastly, the observed infarct volume was 26%(p<0.05), 25%(p<0.05), and 16%(p<0.05) higher in the 0, 15, and 30 day NW groups respectively, when compared to the saline group. Conclusion: Even after 30 days of NW, N-induced global metabolomic changes in the brain persist and may be responsible for increased ischemic brain damage as well as cognitive deficits in female rats.Academi

The Impact of Nicotine along with Oral Contraceptive Exposure on Brain Fatty Acid Metabolism in Female Rats

Smoking-derived nicotine (N) and oral contraceptive (OC) synergistically exacerbate ischemic brain damage in females, and the underlying mechanisms remain elusive. In a previous study, we showed that N + OC exposure altered brain glucose metabolism in females. Since lipid metabolism complements glycolysis, the current study aims to examine the metabolic fingerprint of fatty acids in the brain of female rats exposed to N+/&minus;OC. Adolescent and adult Sprague&ndash;Dawley female rats were randomly (n = 8 per group) exposed to either saline or N (4.5 mg/kg) +/&minus;OC (combined OC or placebo delivered via oral gavage) for 16&ndash;21 days. Following exposure, brain tissue was harvested for unbiased metabolomic analysis (performed by Metabolon Inc., Morrisville, NC, USA) and the metabolomic profile changes were complemented with Western blot analysis of key enzymes in the lipid pathway. Metabolomic data showed significant accumulation of fatty acids and phosphatidylcholine (PC) metabolites in the brain. Adolescent, more so than adult females, exposed to N + OC showed significant increases in carnitine-conjugated fatty acid metabolites compared to saline control animals. These changes in fatty acyl carnitines were accompanied by an increase in a subset of free fatty acids, suggesting elevated fatty acid &beta;-oxidation in the mitochondria to meet energy demand. In support, &beta;-hydroxybutyrate was significantly lower in N + OC exposure groups in adolescent animals, implying a complete shunting of acetyl CoA for energy production via the TCA cycle. The reported changes in fatty acids and PC metabolism due to N + OC could inhibit post-translational palmitoylation of membrane proteins and synaptic vesicle formation, respectively, thus exacerbating ischemic brain damage in female rats

Post-stroke periodic estrogen receptor-beta agonist improves cognition in aged female rats

Women have a higher risk of having an ischemic stroke and increased cognitive decline after stroke as compared to men. The female sex hormone 17β-estradiol (E2) is a potent neuro- and cognitive-protective agent. Periodic E2 or estrogen receptor subtype-beta (ER-β) agonist pre-treatments every 48 h before an ischemic episode ameliorated ischemic brain damage in young ovariectomized or reproductively senescent (RS) aged female rats. The current study aims to investigate the efficacy of post-stroke ER-β agonist treatments in reducing ischemic brain damage and cognitive deficits in RS female rats. Retired breeder (9-10 months) Sprague-Dawley female rats were considered RS after remaining in constant diestrus phase for more than a month. The RS rats were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min and treated with either ER-β agonist (beta 2, 3-bis(4-hydroxyphenyl) propionitrile; DPN; 1 mg/kg; s.c.) or DMSO vehicle at 4.5 h after induction of tMCAO. Subsequently, rats were treated with either ER-β agonist or DMSO vehicle every 48 h for ten injections. Forty-eight hours after the last treatment, animals were tested for contextual fear conditioning to measure post-stroke cognitive outcome. Neurobehavioral testing, infarct volume quantification, and hippocampal neuronal survival were employed to determine severity of stroke. Periodic post-stroke ER-β agonist treatment reduced infarct volume, improved recovery of cognitive capacity by increasing freezing in contextual fear conditioning, and decreased hippocampal neuronal death in RS female rats. These data suggest that periodic post-stroke ER-β agonist treatment to reduce stroke severity and improve post-stroke cognitive outcome in menopausal women has potential for future clinical investigation

Preclinical Evaluation of Safety and Biodistribution of Red Cell Microparticles: A Novel Hemostatic Agent

Uncontrollable bleeding is a major cause of mortality and morbidity worldwide. Effective hemostatic agents are urgently needed. Red cell microparticles (RMPs) are a highly promising hemostatic agent. This study evaluated the safety profile of RMPs preliminary to clinical trial. RMPs were prepared from type O+ human red blood cell by high-pressure extrusion. Male rats were treated with RMPs either a 1 × bolus, or 4 × or 20 × administered over 60 minutes. The vehicle-treated group was used as a control. Effects on physiological parameters were evaluated; namely, blood pressure, body and head temperature, hematocrit, and blood gases. We did not observe any adverse effects of RMPs on these physiological parameters. In addition, brain, heart, and lungs of rats treated with 4 × dose (bolus followed by infusion over 60 minutes) or vehicle were examined histologically for signs of thrombosis or other indications of toxicity. No thrombosis or indications of toxicity in brain, heart, or lungs were observed. Studies revealed that RMPs were distributed mainly in liver, spleen, and lymph nodes, and were potentially excreted through the kidneys. Our study indicates that RMP administration appears not to have any negative impact on the parameters studied and did not produce thrombosis in heart, brain, and lungs. However, more detailed long-term studies confirming the safety of RMP as a hemostatic agent are warranted

Sex-Dependent Differences in Physical Exercise-Mediated Cognitive Recovery Following Middle Cerebral Artery Occlusion in Aged Rats

Stroke remains a leading cause of death and disability in the United States. No current treatments exist to promote cognitive recovery in survivors of stroke. A previous study from our laboratory determined that an acute bout of forced treadmill exercise was able to promote cognitive recovery in 3 month old male rats after middle cerebral artery occlusion (MCAo). In this study, we tested the hypothesis that 6 days of intense acute bout of forced treadmill exercise (physical exercise – PE) promotes cognitive recovery in 11–14 month old male rats. We determined that PE was able to ameliorate cognitive deficits as determined by contextual fear conditioning. Additionally, we also tested the hypothesis that PE promotes cognitive recovery in 11–13 month old reproductive senescent female rats. In contrast to males, the same intensity of exercise that decrease cognitive deficits in males was not able to promote cognitive recovery in female rats. Additionally, we determined that exercise did not lessen infarct volume in both male and female rats. There are many factors that contribute to higher stroke mortality and morbidities in women and thus, future studies will investigate the effects of PE in aged female rats to identify sex differences