Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain

P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, ³H-verapamil (threefold increase), ³H-loperamide (fivefold increase), and ³H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain ¹⁴C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.     

Prognostic impact of high ABC transporter activity in 111 adult acute myeloid leukemia patients with normal cytogenetics when compared to FLT3, NPM1, CEBPA and BAALC

ATP-binding cassette transporter (and specially P-glycoprotein) activity is a well known prognostic factor in acute myeloid leukemia, but when compared to other molecular markers its prognostic value has not been well studied. Here we study relationships between this activity, fms-like tyro-sine kinase 3(FLT3/ITD), nucleophosmin(NPM1), CAAT-enhancer binding protein alpha(CEBPα), and brain and acute leukemia cytoplasmic protein (BAALC), in 111 patients with normal cytogenetics who underwent the same treatment, and evaluate its prognostic impact. Independent factors for survival were age (P=0.0126), ATP-binding cassette transporter activity (P=0.018) and duplications in the fms-like tyrosine kinase 3 (P=0.0273). In the 66 patients without fms-like tyrosine kinase 3 duplication and without nucleophosmin mutation, independent prognostic factors for complete remission achievement and survival were age and ATP-binding cassette transporter activity. In conclusion, ATP-binding cassette transporter activity remains an independent prognostic factor, and could assist treatment decisions in patients with no nucleophosmin mutation and no fms-like tyrosine kinase 3 duplication.     

Essentials of cell physiology

Cell physiology and molecular genetics now provide the basis for the fundamental understanding of the mechanisms involved in normal and pathological physiology, modes of drug action, and risks involved in surgical procedures. Some explanations, often involving a single mutation in a protein are straightforward (e.g. cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis). Others may involve a constellation of effects (e.g. obesity) and surprisingly there still remain important areas lacking comprehensive explanation (e.g. the mode of action of general anaesthetics) or close to resolution but still lacking detail (aldosterone controlling both K and Na regulation in the kidney). The present article presents a concise summary of regulation of cell function, concentrating on the cell membrane and important organelles. Signalling and second messengers, together with the role of membrane channels and transporters constitute an important aspect of this, mediated via kinases and phosphatases. Cell volume regulation, reflecting swelling and oedema, are an important aspect of organ transplantation and brain function.     

宁夏回族脑梗死患者三磷酸腺苷结合盒转运子基因R219K多态性与血脂的关系

目的探讨宁夏回族脑梗死患者ATP结合盒转运子(ABCA1)基因R219K多态性与血脂的关系。方法连续入选住院的回族脑梗死患者105例为脑梗死组,同期入选我院门诊回族健康体验者257例为对照组。检测血脂等生化指标,用PCR-RFLP方法测基因R219K多态性,分析其与血脂的关系。结果脑梗死组和对照组RR、RK基因型及R和K等位基因频率比较,差异无统计学意义(P>0.05)。脑梗死组KK基因型较对照组明显降低,差异有统计学意义(11.4%vs 26.8%,P<0.01)。各基因型血脂水平比较,差异无统计学意义(P>0.05)。结论 KK基因型可能是回族脑梗死患者的保护因素,其保护因素可能与血脂水平无关。     

高糖对THP-1巨噬细胞ABC转运体的表达和功能的影响

目的:探讨高糖对体外培养的THP-1巨噬细胞中三磷酸腺苷结合盒（ABC）转运体的表达及功能的影响。方法: 以不同浓度的D-葡萄糖干预培养的THP-1单核巨噬细胞5 d,用实时定量PCR和Western blot检测巨噬细胞中ABCG1、ABCA1 mRNA和其蛋白的表达。用酶荧光化学法检测培养基中及细胞内胆固醇的含量。结果: 高糖可抑制巨噬细胞中ABCG1的表达,但是对ABCA1的表达影响不明显。随着D-葡萄糖浓度的增加,从巨噬细胞中流出的胆固醇量减少,同时细胞内胆固醇的含量增加。结论: 高糖可抑制巨噬细胞中ABCG1的表达及功能,有助于促进巨噬细胞内脂质堆积。     

硫酸软骨素蛋白多糖与中枢神经系统可塑性

硫酸软骨素蛋白多糖(chondroitin sulfate proteoglycan,CSPG)是在发育和成熟的中枢神经系统(central nervous syste,CNS)中广泛表达的一组细胞外基质分子,在胚胎CNS发育和成年期CNS可塑性中发挥着重要作用.CSPG作为一种主要的细胞外抑制性成分,可影响CNS损伤后轴突再生和神经功能恢复.     

Effect of chondroitinase ABC on adhesion and behavior of synovial membrane‐derived mesenchymal stem cells in rabbit partial‐thickness chondral defects

Transplanted cells may have difficulty attaching to the surface of partial‐thickness chondral lesions because of the anti‐adhesive properties of the proteoglycan rich matrix. Therefore, the current study attempts to evaluate the effect of chondroitinase ABC (chABC) on the adhesion and behavior of transplanted synovial membrane‐derived mesenchymal stem cells (SDSCs) in rabbit partial‐thickness chondral defects. In ex vivo adhesion experiments, chABC treatment (0.1 U/ml) was increased in SDSC attachment to the cartilage explants, and significantly diminished by pretreatment with neutralizing antibody against fibronectin. In the in vivo experiments, 1 day and 4 weeks after the chABC treatment (0.1 and 1 U/ml), the immunoreactivity (IR) against CS‐56 (intact chondroitin sulfate antibody) was markedly decreased; however, the IR of 2B6 (stub of the chondroitin 4‐sulfate chain), 3B3 (stub of the chondroitin 6‐sulfate chain), and fibronectin was increased. At 12 weeks, this IR returned to normal except in the high‐dose chABC‐treated group (1 U/ml). Furthermore, the attachment of SDSCs to the chondral defects after chABC treatment was increased at 7 days compared with that in the chondral defects pretreated with saline. However, the tissue repaired by SDSCs was negatively stained for type II collagen at 12 weeks. In conclusion, these results showed that the exposure to fibronectin by chABC treatment enhances the attachment of SDSCs to partial‐thickness chondral defects. However, the tissue regenerated by SDSCs showed lack of hyaline cartilage regeneration. Thus, to understand the fate of transplanted MSCs in cartilage defect is very important for successful cell therapies. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1293–1301, 2013     

A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility

Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell‐biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1^?/? mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high‐pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde‐induced flattening of SVs observed in VGLUT1^?/? synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1^?/? presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1^?/? mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2–enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1^?/? neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals.     

药物转运体表观遗传调控机制的研究进展

药物转运体在体内药物吸收、分布和排泄过程中发挥着重要的作用。转运体在各组织器官的分布和表达受到表观遗传修饰调控,导致某些药物体内处置过程出现明显的个体差异。随着表观遗传学的发展,基于表观遗传修饰（如DNA甲基化、组蛋白修饰、microRNA干预等）调控药物转运体的相关研究越来越多。对表观遗传调控药物转运体研究进行综述。     

Advances and challenges in hereditary cancer pharmacogenetics

Introduction: Cancer pharmacogenetics usually considers tumor-specific targets. However, hereditary genetic variants may interfere with the pharmacokinetics of antimetabolites and other anti-cancer drugs, which may lead to severe adverse events.

Areas covered: Here, the impact of hereditary genes considered in drug labels such as thiopurine S-methyltransferase (TPMT), UDP-glucuronosyltransferase 1A1 (UTG1A1) and dihydropyrimidine dehydrogenase (DPYD) are discussed with respect to guidelines of the Clinical Pharmacogenetics Implementation Consortium (CPIC). Moreover, the association between genetic variants of drug transporters with the clinical outcome is comprehensively discussed.

Expert opinion: Precision therapy in the field of oncology is developing tremendously. There are a number of somatic tumor genetic markers that are indicative for treatment with anti-cancer drugs. By contrast, for some hereditary variants, recommendations have been developed. Although we have vast knowledge on the association between drug transporter variants and clinical outcome, the overall data is inconsistent and the predictability of the related phenotype is low. Further developments in research may lead to the discovery of rare, but functionally relevant single nucleotide polymorphisms and a better understanding of multiple genomic, epigenomic as well as phenotypic factors, contributing to drug response in malignancies.