Interaction between CFTR and prestin (SLC26A5)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin-CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP-activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.     

Lipid-based colloidal carriers for peptide and protein delivery--liposomes versus lipid nanoparticles

This paper highlights the importance of lipid-based colloidal carriers and their pharmaceutical implications in the delivery of peptides and proteins for oral and parenteral administration. There are several examples of biomacromolecules used nowadays in the therapeutics, which are promising candidates to be delivered by means of liposomes and lipid nanoparticles, such as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). Several production procedures can be applied to achieve a high association efficiency between the bioactives and the carrier, depending on the physicochemical properties of both, as well as on the production procedure applied. Generally, this can lead to improved bioavailability, or in case of oral administration a more consistent temporal profile of absorption from the gastrointestinal tract. Advantages and drawbacks of such colloidal carriers are also pointed out. This article describes strategies used for formulation of peptides and proteins, methods used for assessment of association efficiency and practical considerations regarding the toxicological concerns.     

Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes

In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.     

长春新碱阳离子纳米结构脂质载体及其小肠吸收的研究

目的:硫酸长春新碱作为一种细胞毒型抗肿瘤药物,临床上多用其注射剂,虽应用广泛,但存在较多缺点,如药物半衰期短,代谢速率快以及毒副作用明显。本文目的是制备包载长春新碱和十二烷基磺酸钠的阳离子纳米结构脂质载体,并对其进行评价。方法:用复乳挥发法制备出目标脂质纳米粒;利用激光粒度仪对其粒径及zeta电位进行检测;利用高效液相色谱法对其包封率和载药量进行测定;透析法检测纳米粒的体外释放行为;用小肠吸收法评价纳米粒的促进吸收作用。结果:制得的纳米粒的平均粒径为(192.4±4.14)nm,多分散系数(PDI)为0.184±0.015,包封率为32.28%,Zeta电位为(30.6±4.09)m V,载药量为(1.56±0.10)%;体外释放实验显示在pH=7.4的中性释放介质中,硫酸长春新碱脂质纳米粒表现出缓释特性;小肠吸收实验表明十二烷基磺酸钠的加入和阳离子纳米粒的修饰可提高小肠对药物的吸收。结论:阳离子硫酸长春新碱纳米结构脂质载体具有缓释效果,并可以促进小肠对药物的吸收。     

Evaluation of alternative strategies to optimize ketorolac transdermal delivery

In the present study, 2 alternative strategies to optimize ketorolac transdermal delivery, namely, prodrugs (polyoxyethylene glycol ester derivatives, I–IV) and nanostructured lipid carriers (NLC) were investigated. The synthesized prodrugs were chemically stable and easily degraded to the parent drug in human plasma. Ketorolac-loaded NLC with high drug content could be successfully prepared. The obtained products formulated into gels showed a different trend of drug permeation through human stratum corneum and epidermis. Particularly, skin permeation of ester prodrugs was significantly enhanced, apart from ester IV, compared with ketorolac, while the results of drug release from NLC outlined that these carriers were ineffective in increasing ketorolac percutaneous absorption owing to a higher degree of mutual interaction between the drug and carrier lipid matrix. Polyoxyethylene glycol esterification confirmed to be a suitable approach to enhance ketorolac transdermal delivery, while NLC seemed more appropriate for sustained release owing to the possible formation of a drug reservoir into the skin. Published: August 4, 2006     

固体脂质纳米粒的制备及在治疗中的应用研究

固体脂质纳米粒(SLN)是20世纪90年代发展起来的一种性能优异的新型纳米粒给药剂型作为一种新型载体,可有效提高包封药物的稳定性、提高病变部位靶向性、低毒性与组织亲和性,为药物的体内递送提供了一种新的方法。本文主要针对固体脂质纳米粒的制备,发展现状,目前存在的问题及解决思路等作以介绍与总结。并在此基础上,介绍了新的脂质纳米粒,纳米脂质载体(nanostructured lipid carriers,NLC)和药脂结合物纳米粒(Lipid drug conjugate nanoparticles,LDC),以及未来固体脂质纳米粒的发展方向。     

Structure of the Cytosolic Portion of the Motor Protein Prestin and Functional Role of the STAS Domain in SLC26/SulP Anion Transporters

Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti-sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti-sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 Å resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which—although previously considered merely as a generic linker between the domain and the last transmembrane helix—is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties.     

Mutation-induced reinforcement of prestin-expressing cells

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.     

Salicylate-induced translocation of prestin having mutation in the GTSRH sequence to the plasma membrane

Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin-transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state.     

Evaluation of the in vitro differential protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (NLCs) for potential targeting to the brain

The preferential in vitro adsorption of apolipoprotein E (Apo E) onto the surface of colloidal drug carriers may be used as a strategy to evaluate the in vivo potential for such systems to transport drugs to the brain. The aim of this research was to investigate the in vitro protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (DDI-NLCs), using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), in order to establish the potential for NLCs to deliver DDI to the brain. NLC formulations were manufactured using high-pressure homogenization using a lipid matrix consisting of a mixture of Precirol^® ATO 5 and Transcutol^® HP. The 2-D PAGE analysis revealed that NLCs in formulations stabilized using Solutol^® HS 15 alone or with a ternary surfactant system consisting of Solutol^® HS 15, Tween^® 80, and Lutrol^® F68, preferentially adsorbed proteins, such as Apo E. Particles stabilized with Tween^® 80 and Lutrol^® F68 did not adsorb Apo E in these studies, which could be related to the relatively large particle size and hence small surface area observed for these NLCs. These findings have revealed that DDI-loaded NLCs may have the potential to deliver DDI to the brain in vivo and, in addition, to Tween^® 80, which has already been shown to have the ability to facilitate the targeting of colloidal drug delivery systems to the brain. Solutol^® HS 15–stabilized nanoparticles may also achieve a similar purpose.