Effect of a non-methoxylated N-trimethyl chitosan chloride derivative over capreomycin sulfate permeability in CaCo-2 cell monolayers

The effect of N-trimethylchitosan chloride on the intestinal permeability of capreomycin sulfate, a polypeptide antibiotic, using an in vitro model, was evaluated. To improve the mucoadhesivity and permeation enhancer properties of N-trimethylchitosan chloride, it was used a synthetic pathway that selectively alkylated the amino groups and not the hydroxyl groups in carbons 3 and 6, which decreases the potency of the polymer, leading to use higher quantities and limit its potential as a functional excipient. This non-methoxylated derivative of the studied polymer reduced in a reversible way the transepithelial electrical resistance of CaCo-2 monolayers at concentrations not higher than 0.003 % w/v, indicating that the absence of steric hindrance from methoxyl groups improves the effect of N-trimethylchitosan chloride, but at expense of a narrow range of action and higher cytotoxicity. The results of in vitro permeation studies conducted in bicameral Transwell® systems suggested that the permeability of capreomycin sulfate is low, although it was not established if the transport is exclusively paracellular or membrane transporters are involved. By using increasing concentrations of the polymer in the range of 0.001 - 0.003% w/v, it was observed a slight increase in the transport of capreomycin. Therefore, it was concluded that further studies should use N-trimethylchitosan chloride with a lower degree of quaternization or controlled methoxylation, in order to increase the mass to use and obtain a product that is able to remain retained in the intestinal lumen and which in turn interacts with the epithelium.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways

Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A analog) or hydrogen peroxide (HO) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by HO and U46619 was attenuated and the effect on HO-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This phenomenon is related to quick increases of hCAT-1 abundance and higher capacity of endothelial cells to take up L-arginine and generate NO

Heat-stable microtubule protein MAP-1 binds to microtubules and induces microtubule assembly

AbstractThe microtubule-associated proteins, MAP-1, MAP-2 and tau, have been purified from brain tissue via a new approach using a heating step directly on the homogenate, followed by selective adsorption on calmodulin-Sepharose affinity columns and gel-filtration chromatography. Our results indicate that these MAPs share common biochemical properties, including heat stability, calmodulin binding and promotion of tubulin assembly into microtubules

Identification of Structural Determinants of the Transport of the Dehydroascorbic Acid Mediated by Glucose Transport GLUT1

GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1’s transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the Vmax compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C’s transport via GLUT1

PrPC as a Transducer of Physiological and Pathological Signals

International audienceAfter the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrPC) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrPC in health and disease. PrPC, which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrPC remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrPC, its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrPC an interesting pharmacological target. In a physiological context, several reports have proposed that PrPC modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrPC has also been implicated in the pathophysiological cell signaling induced by β-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer’s disease (AD), as a mediator of Aβ-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrPC as a transducer of physiological and pathological signaling. Copyrigh

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein

Insulin regulates the L-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the L-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on L-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced L-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or N-nitro-L-arginine methyl ester. L-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases L-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated L-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of L-arginine transport in HUVECs