Increased complexity of vasopressin's vascular actions

Vasopressin is becoming a widely used pressor in conditions with severe hypotension. Like several other hormones important in cardiovascular and extracellular fluid control, however, vasopressin can activate several receptors that when pharmacologically or pathologically stimulated may result in conflicting effects. In the present issue of Critical Care, Rehberg and colleagues examined the hypothesis that blockade of vasopressin V2 receptor during septic shock may be beneficial. Their tantalizing results indicate that future work must consider the precise vasopressin receptors that are stimulated and/or inhibited

Targeting the NO/cGMP/CREB Phosphorylation Signaling Pathway in Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and the most common form of senile dementia. Recently, scientists have put significant effort into exploring the molecular mechanisms involved in the pathological processes leading to the disease. A vast number of studies have focused on understanding the nitric oxide (NO) signaling pathway, which culminates with the phosphorylation of the transcription factor cAMP-responsive element-binding protein (CREB) through the increase of the second messenger cyclic guanosine monophosphate (cGMP) and activation of cGMP-dependent protein kinase. This book chapter provides an overview of the progress being made in modulating the hippocampal synaptic transmissions, which are critical for learning and memory, by targeting the different components of the NO/cGMP/CREB phosphorylation signaling pathway. Furthermore, a description of recent research on this pathway through the use of phosphodiesterase inhibitors is emphasized

Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

We report the derivation and characterization of two new human embryonic stem cells (hESC) lines (CU1 and CU2) from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED) 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF) embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos

Position and Role of the BK Channel α Subunit S0 Helix Inferred from Disulfide Crosslinking

The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage- and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfide bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular flank of S0 is surrounded on three sides by the extracellular flanks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine–substituted alphas, each with one cysteine in the S0 flank and one in the S3–S4 loop, were at least 90% disulfide cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V50 or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charge bearing S3 and S4 could contribute to the considerably larger electrostatic energy required to activate the BK channel compared with typical voltage-gated potassium channels with six TM helices

Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability

Anti-Cocaine Compositions and Treatment

Embodiments of the invention disclosed herein generally relate to anti-cocaine therapeutics. Specifically, some embodiments of the invention relate to highly efficient, thermostable, and long-lasting cocaine esterase (CocE) mutants that can protect against the toxic and reinforcing effects of cocaine in subjects. Provided herein are mutant CocE polypeptides displaying thermostable esterase activity. Also provided are methods of treating cocaine-induced conditions in a subject in need via administration of mutant CocE as well as methods for high-throughput screening of candidate esterase polypeptides