BACKGROUND & AIMS: PURPOSE:A phase II trial of Photofrin-mediated i.p. photodynamic therapy shown in a previous report limited efficacy and significant acute, but not chronic, toxicity. A secondary aim of this trial and the subject of this report is to determine Photofrin uptake in tumor and normal tissues. EXPERIMENTAL DESIGN:Patients received Photofrin, 2.5 mg/kg, i.v., 48 hours before debulking surgery. Photofrin uptake was measured by spectroflurometric analysis of drug extracted from tumor and normal tissues removed at surgery. Differences in drug uptake among these tissues were statistically considered using mixed-effects models. RESULTS:Photofrin concentration was measured in 301 samples collected from 58 of 100 patients enrolled on the trial. In normal tissues, drug uptake significantly (P<0.0001) differed as a function of seven different tissue types. In the toxicity-limiting tissue of intestine, the model-based mean (SE) Photofrin level was 2.70 ng/mg (0.32 ng/mg) and 3.42 ng/mg (0.24 ng/mg) in full-thickness large and small intestine, respectively. In tumors, drug uptake significantly (P=0.0015) differed as a function of patient cohort: model-based mean Photofrin level was 3.32 to 5.31 ng/mg among patients with ovarian, gastric, or small bowel cancer; 2.09 to 2.45 ng/mg among patients with sarcoma and appendiceal or colon cancer; and 0.93 ng/mg in patients with pseudomyxoma. Ovarian, gastric, and small bowel cancers showed significantly higher Photofrin uptake than full-thickness large and/or small intestine. However, the ratio of mean drug level in tumor versus intestine was modest (

BACKGROUND & AIMS: :The final elimination step of self-reactive T cells occurs in the medulla of the thymus where a complex framework provided by stromal cells supports an optimal milieu for their selection. Here we present evidence that tight junctions (TJs) widely join medullary stromal cells of the human thymus. Occludin (OCLN) and claudin-1 (CLDN-1) of TJ-associated molecules were dominantly expressed in medullary thymic epithelial cells (mTECs), and CLDN-4 and CLDN-7 were also localized in some mTECs near Hassall's corpuscles. Interestingly, p53-like transcription factors were found to upregulate OCLN and CLDN-1 in human TEC lines, as recently suggested in the regulation of mTEC function. Furthermore, dendritic cells (DCs) of the medulla, with a major role for selection of thymocytes, expressed CLDN-1 and OCLN as well, implying that the interposition of DCs within the mTEC scaffold is also helped by TJs. Analysis of freeze-fracture replicas of the thymus revealed TJ strand structures in the vicinity of gap junction plaques through which small molecules might move, as implied by dye-transfer analysis of a medullary cell line. Thus, it is thought that p53-like molecules regulate TJ-associated interactions of medullary stromal cells and that this mechanism might be associated with an intercellular communication network, probably for preserving the medullary niches.

背景与目标： ：自我反应性T细胞的最终消除步骤发生在胸腺的髓质中，其中基质细胞提供的复杂框架为它们的选择提供了最佳环境。在这里，我们提供证据表明紧密连接（TJs）广泛加入人类胸腺的髓质基质细胞。 TJ相关分子的Occludin（OCLN）和claudin-1（CLDN-1）主要在髓样胸腺上皮细胞（mTECs）中表达，CLDN-4和CLDN-7也位于Hassall小体附近的某些mTECs中。有趣的是，正如最近在调控mTEC功能中所建议的那样，发现p53样转录因子可上调人TEC细胞系中的OCLN和CLDN-1。此外，在选择胸腺细胞中起主要作用的髓质树突状细胞（DC）也表达CLDN-1和OCLN，这暗示TJ也有助于mTEC支架内DC的插入。胸腺的冷冻断裂复制品的分析显示，在间隙连接斑块附近的TJ链结构中，小分子可能会通过间隙连接斑块移动，正如髓细胞系的染料转移分析所暗示的那样。因此，认为p53样分子调节髓质基质细胞的TJ相关相互作用，并且该机制可能与细胞间通讯网络有关，可能是为了保留髓ni。

BACKGROUND & AIMS: :Rhodopseudomonas palustris is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The R. palustris genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the R. palustris regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in R. palustris cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in R. palustris.

背景与目标： ：Rhodopseudomonas palustris是一种紫色的兼性光养细菌，它利用氢气作为电子供体，在光养植物生长过程中固定二氧化碳，或在固氮过程中合成氨气。它还使用氢作为电子补充剂，以在光异养生长期间将氧化的碳化合物（例如苹果酸）完全同化到细胞材料中。 R. palustris基因组预测膜结合的镍铁摄取氢化酶和几种调节蛋白来控制氢化酶的合成。与氢化酶基因簇直接相邻的还有一个新的传感器激酶基因（RPA0981）。在这里，我们显示帕氏疟原虫调节传感器氢化酶HupUV与传感器激酶响应调节蛋白对HoxJ-HoxA共同作用，以响应氢气激活氢化酶表达。转录组分析表明，HupUV-HoxJA调节系统还控制编码预测的二羧酸转运系统，推定的甲酸盐转运蛋白和谷氨酰胺合成酶的基因的表达。 RPA0981在抑制氢化酶合成方面作用很小。我们还确定了两组分系统RegS-RegR抑制摄取氢化酶的表达，可能是响应细胞内氧化还原状态的变化。转录组分析表明，与缺乏摄取氢酶的突变菌株相比，当在固氮条件下在苹果酸上光异养生长时，利用氢的pal.ris细胞中约有30个基因差异表达。由此看来，还原剂以氢的形式的循环利用对R. palustris的基因表达没有广泛的非特异性影响。

BACKGROUND & AIMS: :Professional antigen-presenting cells (APC) are able to process and present exogenous antigen leading to the activation of T cells. Antigen-immunoglobulin (Ig)G complexes (IC) are much more efficiently processed and presented than soluble antigen. Dendritic cells (DC) are known for their ability to take up and process immune complex (IC) via FcgammaR, and they have been shown to play a crucial role in IC-processing onto major histocompatibility complex (MHC) class I as they contain a specialized cross-presenting transport system required for MHC class I antigen-processing. However, the MHC class II-antigen-processing pathway is distinct. Therefore various other professional APC, like macrophages and B cells, all displaying FcgammaR, are thought to present IC-delivered antigen in MHC class II. Nonetheless, the relative contribution of these APC in IC-facilitated antigen-presentation for MHC class II in vivo is not known. Here we show that, in mice, both macrophages and DC, but not B cells, efficiently capture IC. However, only DC, but not macrophages, efficiently activate antigen-specific MHC class II restricted CD4(+) T cells. These results indicate that mainly DC and not other professional APC, despite expressing FcgammaR and MHC class II, contribute significantly to IC-facilitated T cell activation in vivo under steady-state conditions.

背景与目标： ：专业抗原呈递细胞（APC）能够处理并呈递导致T细胞活化的外源性抗原。抗原-免疫球蛋白（Ig）G复合物（IC）比可溶性抗原的加工和呈递效率更高。树突状细胞（DC）以通过FcgRR吸收和加工免疫复合物（IC）的能力而闻名，并且由于它们含有I型树突状细胞（DC），因此它们在I类加工主要组织相容性复合物（MHC）的过程中起着至关重要的作用。 MHC I类抗原加工所需的专业交叉展示转运系统。但是，MHC II类抗原加工途径是不同的。因此，各种其他的专业APC，如巨噬细胞和B细胞，都显示FcgR，被认为在II类MHC中呈递了IC递送的抗原。然而，尚不清楚这些APC在体内II型MHC的IC促进的抗原呈递中的相对贡献。在这里，我们表明，在小鼠中，巨噬细胞和DC都有效捕获了IC，而B细胞却没有。但是，只有DC，而不是巨噬细胞，可以有效地激活II型MHC限制性抗原特异性CD4（）T细胞。这些结果表明，尽管表达FcgammaR和II类MHC，主要还是DC，而不是其他专业APC，在稳态条件下在体内促进了IC促进的T细胞活化。

BACKGROUND & AIMS: :An immediately inducible gene by gonadotropin was isolated from rat ovaries primed with pregnant mare serum gonadotropin (PMSG) by using a subtraction cloning procedure. Homology analysis revealed that the gene is a rat homologue of scavenger receptor class B-I, which was recently identified as a specific receptor for high density lipoprotein (HDL). The structure of rat SRBI was determined by nucleotide sequence analysis of full-length cDNAs for SRBI. Northern blot analysis revealed that rat SRBI mRNA levels were rapidly and strongly increased within 3 h by the injection of PMSG. In situ hybridization study revealed that SRBI mRNA was strongly induced in theca interna cells of immature rat ovary stimulated with 30 IU of PMSG for 6 h. SRBI mRNA expression was also observed in corpora lutea of the adult rat ovary. These findings indicate that expression of SRBI mRNA is restricted to and induced in the ovarian steroidogenic cell types where cholesterol is used as a substrate for synthesis of steroid hormones. Our data strongly suggest that SRBI may play a significant role in the ovarian steroidogenesis by mediating selective uptake of cholesterol from HDL to ovarian theca interna cells or to corpus luteum.

背景与目标： ：通过减法克隆程序，从用怀孕母马血清促性腺激素（PMSG）引发的大鼠卵巢中分离出由促性腺激素立即诱导的基因。同源性分析显示该基因是清道夫受体B-I类的大鼠同源物，最近被鉴定为高密度脂蛋白（HDL）的特异性受体。通过SRBI全长cDNA的核苷酸序列分析确定大鼠SRBI的结构。 Northern印迹分析表明，通过注射PMSG，大鼠SRBI mRNA水平在3小时内迅速而强烈地增加。原位杂交研究表明，SRBI mRNA在30 IU PMSG刺激6 h的未成熟大鼠卵巢的内膜细胞中被强烈诱导。在成年大鼠卵巢的黄体中也观察到了SRBI mRNA表达。这些发现表明，SRBI mRNA的表达受限于卵巢类固醇生成细胞类型并在其中被诱导，其中胆固醇被用作类固醇激素合成的底物。我们的数据有力地表明，SRBI可能通过介导HDL对胆固醇和卵巢黄体的选择性摄取来介导胆固醇的选择性摄取，从而在卵巢类固醇生成中发挥重要作用。

BACKGROUND & AIMS: :Adenovirus infection results in the suppression of cellular protein synthesis, but the mechanism has not been established. In this report we demonstrate that the shut-off of cellular protein synthesis by adenovirus is prevented in cells by treatment with the drug 2-aminopurine. Treatment with 2-aminopurine is shown to prevent suppression of cellular translation without disrupting the normal viral block in the transport of cellular mRNAs from the nucleus to the cytoplasm. We show that viral suppression of cellular protein synthesis occurs concomitant with activation of the interferon-induced double-stranded RNA-activated inhibitor (DAI), a protein kinase, and phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), but that prevention of host cell shut-off by 2-aminopurine occurs without a decrease in kinase activity or a dephosphorylation of eIF-2 alpha. Results are presented that indicate that activation of DAI kinase and phosphorylation of eIF-2 alpha may be required but are not sufficient to achieve inhibition of cellular protein synthesis during adenovirus infection. We suggest that other events, in particular the modification of additional initiation factors, are likely involved in viral inhibition of cellular translation.

背景与目标： ：腺病毒感染导致细胞蛋白质合成受到抑制，但该机制尚未建立。在本报告中，我们证明了通过用2-氨基嘌呤药物治疗可防止腺病毒关闭细胞蛋白质合成。已显示用2-氨基嘌呤处理可防止细胞翻译受到抑制，而不会破坏细胞mRNA从细胞核到细胞质的运输过程中的正常病毒阻滞。我们显示病毒抑制细胞蛋白质合成的发生与干扰素诱导的双链RNA激活的抑制剂（DAI），蛋白激酶和真核生物起始因子2（eIF-2 alpha）的α亚基的磷酸化的激活同时发生。 ，但可以防止2-氨基嘌呤阻止宿主细胞的关闭，而不会降低激酶活性或eIF-2α的去磷酸化。结果表明，可能需要DAI激酶的激活和eIF-2α的磷酸化，但不足以抑制腺病毒感染期间细胞蛋白质合成。我们建议其他事件，特别是其他起始因子的修饰，可能与细胞翻译的病毒抑制有关。

BACKGROUND & AIMS: BACKGROUND:The major clinical feature of ataxia telangiectasia (A-T) is severe progressive neurodegeneration with onset in infancy. This classical A-T phenotype is caused by biallelic null mutations in the ATM gene, leading to the absence of ATM protein and increased cellular radiosensitivity. We report an unusual case of A-T in a 41-year-old mother, A-T210, who had very mild neurological symptoms despite complete loss of ATM protein. METHODS:A neurological examination was performed, cellular radiosensitivity was assessed, and the ATM gene was sequenced. Skin fibroblasts and a lymphoblastoid cell line (LCL) were assayed for ATM protein expression and kinase activity. RESULTS:Patient A-T210 showed mild chorea, dystonia, and gait ataxia, walked independently, and drove a car. LCL and skin fibroblasts were radiosensitive and did not express ATM protein. Two ATM-null mutations were identified. CONCLUSIONS:The severe neurodegeneration resulting from loss of ATM can be mitigated in some circumstances.

背景与目标： 摘要背景：共济失调性毛细血管扩张症（A-T）的主要临床特征是严重的进行性神经变性，并在婴儿期发作。这种经典的A-T表型是由ATM基因中的双等位基因无效突变引起的，从而导致ATM蛋白的缺失和细胞放射敏感性的提高。我们报告了一个41岁母亲A-T210中的A-T异常病例，尽管ATM蛋白完全丧失，但她的神经系统症状非常轻微。
方法：进行神经系统检查，评估细胞放射敏感性，并对ATM基因进行测序。分析了皮肤成纤维细胞和类淋巴母细胞系（LCL）的ATM蛋白表达和激酶活性。
结果：患者A-T210表现为轻度舞蹈症，肌张力障碍和步态共济失调，独立行走并开车。 LCL和皮肤成纤维细胞对放射敏感，不表达ATM蛋白。确定了两个ATM空突变。
结论：在某些情况下，可以缓解ATM丢失导致的严重神经变性。

BACKGROUND & AIMS: AIM:To report the patterns and sites of 18-FDG uptake in patients of presumed ocular tuberculosis. MATERIALS AND METHODS:The clinical and investigational findings of 11 patients were reviewed retrospectively. These included 6 males and 5 females with a mean age of 46.2 years. 21 eyes were included in the data analysis. Clinical presentations include 15 eyes with anterior uveitis, 2 eyes with retinal vasculitis, 2 eyes with panuveitis and 2 eyes with multifocal choroidopathy. RESULTS:Two distinct patterns of systemic uptake emerged. Pattern 1: No detectable systemic uptake (4 patients). Pattern 2: Detectable systemic uptake. a. Chest disease only (2 patients). b. Disseminated pattern, uptake seen at multiple sites (4 patients). c. Extrapulmonary only (1 patient). CONCLUSIONS:Ocular tuberculosis may often be part of a wider disseminated disease.

背景与目标： 目的：报告推测的眼结核患者摄取18-FDG的方式和部位。
材料与方法：回顾性分析11例患者的临床和研究结果。其中包括6名男性和5名女性，平均年龄为46.2岁。数据分析包括21只眼睛。临床表现包括前葡萄膜炎15眼，视网膜血管炎2眼，胰腺炎2眼和多灶性脉络膜病变2眼。
结果：出现了两种不同的全身吸收模式。模式1：未检测到全身吸收（4例患者）。模式2：可检测到的全身吸收。一种。仅胸部疾病（2例）。 b。分布模式，在多个部位可见吸收（4例）。 C。仅肺外（1例患者）。
结论：眼结核通常可能是更广泛传播的疾病的一部分。

BACKGROUND & AIMS: :Cellular iron homeostasis is maintained by iron and heme transport proteins that work in concert with ferrireductases, ferroxidases, and chaperones to direct the movement of iron into, within, and out of cells. Systemic iron homeostasis is regulated by the liver-derived peptide hormone, hepcidin. The interface between cellular and systemic iron homeostasis is readily observed in the highly dynamic iron handling of four main cell types: duodenal enterocytes, erythrocyte precursors, macrophages, and hepatocytes. This review provides an overview of how these cell types handle iron, highlighting how iron and heme transporters mediate the exchange and distribution of body iron in health and disease.

背景与目标： ：铁和血红素转运蛋白与铁还原酶，铁氧化酶和伴侣蛋白协同作用，维持细胞内铁稳态，从而指导铁进入，进入和离开细胞的运动。系统性铁稳态由肝脏衍生的肽激素hepcidin调节。在铁的四种主要细胞类型的高动态铁处理中，很容易观察到细胞与全身铁稳态之间的界面：十二指肠肠上皮细胞，红细胞前体，巨噬细胞和肝细胞。这篇综述概述了这些细胞类型如何处理铁，着重介绍了铁和血红素转运蛋白如何在健康和疾病中介导体内铁的交换和分布。

BACKGROUND & AIMS: :Cervical screening attendance among women aged 25-29 years in England is lower than at older ages. There is some evidence that pre-notification leaflets motivate women who have not yet considered their response to a health intervention. We aimed to identify key information to motivate young women at their first cervical screening invitation. Six focus groups were conducted, five with young women aged 17-25 registered with a General Practice in Manchester, UK, and one with Practice nurses. Some women took part in two further groups to discuss leaflet design. There was low awareness of the purpose or procedures of cervical screening, and most women were de-motivated by reports of bad experiences. Some intended to be screened, but not immediately after invitation. Screening was viewed as a test for a cancer that affected older women. Since none of the participants believed that they had cervical cancer, screening seemed unnecessary. We conclude that the perception that screening is unimportant when you are young needs to be challenged. Women also need to be better informed of screening procedures. A pre-notification leaflet incorporating key information was designed and will be tested in a randomized trial of complex interventions within the routine cervical screening programme.

背景与目标： ：在英格兰，年龄在25-29岁之间的女性接受子宫颈筛查的比例低于年龄较大的女性。有证据表明，预先通知传单可以激励尚未考虑其对健康干预措施反应的妇女。我们旨在识别关键信息，以激发年轻女性的首次宫颈筛查邀请。进行了六个焦点小组讨论，其中五个在17-21岁的年轻女性中注册了英国曼彻斯特的General Practice，一个在执业护士中进行。一些妇女参加了另外两个小组，讨论传单设计。人们对宫颈筛查的目的或程序认识不足，大多数妇女因不良经历的报道而失去动力。有些打算放映，但不是在邀请后立即放映。筛查被认为是对影响老年妇女的癌症的测试。由于没有一个参与者相信自己患有宫颈癌，因此筛查似乎是不必要的。我们得出的结论是，对年轻时进行筛查并不重要的看法需要受到挑战。还需要使妇女更好地了解筛查程序。设计了包含关键信息的预告传单，并将在常规宫颈筛查计划内的复杂干预措施的随机试验中进行测试。

BACKGROUND & AIMS: :Suspension cells and cotyledons of Ricinus communis were compared as to their uptake properties for sugar and amino acids to reveal whether the previously reported sucrose-specificity of the cotyledon is a specific feature of the cotyledon or of the Ricinus cell in general. The experiments show that suspension cells have a higher hexose uptake activity at low sugar concentration than cotyledons, whereas sucrose cannot be taken up by suspension cells unless it is first hydrolyzed. Amino acids are taken up by suspension cells and by the cotyledons. It is concluded that the highly specific uptake of sucrose without hydrolysis is a special feature of certain specialized cells of the cotyledon, probably the phloem.

背景与目标： ：比较了蓖麻的悬浮细胞和子叶的糖和氨基酸摄取特性，以揭示先前报道的子叶的蔗糖特异性是子叶还是一般的蓖麻细胞的特定特征。实验表明，悬浮细胞在低糖浓度下具有比子叶更高的己糖摄取活性，而蔗糖除非先水解就不能被悬浮细胞吸收。氨基酸被悬浮细胞和子叶吸收。结论是，子叶的某些特化细胞（可能是韧皮部）的高度特异摄取不水解的蔗糖是其特殊特征。

BACKGROUND & AIMS: :Atrial fibrillation (AF) is the most frequent arrhythmia and is associated with increased morbidity and mortality. Current drugs for AF treatment have limited efficacy and a substantial risk of proarrhythmic side effects, making novel drug development critical. Emerging evidence suggests that abnormal intracellular calcium (Ca(2+)) signalling is a key contributor to ectopic (triggered) electrical activity in human AF. Accordingly, atrial Ca(2+)-handling abnormalities underlying ectopic activity may constitute novel mechanism-based therapeutic approaches to treat AF. This article reviews the recent evidence for a role of cellular ectopic activity in human AF pathophysiology, discusses the molecular mechanisms underlying triggered activity in human atrial myocytes, and considers their relevance to the design of novel therapeutic options.

背景与目标： ：房颤（AF）是最常见的心律不齐，并与发病率和死亡率增加相关。当前用于AF治疗的药物具有有限的功效和存在心律不齐副作用的巨大风险，这使得新药开发变得至关重要。新兴证据表明，异常的细胞内钙（Ca（2））信号传导是人类房颤异位（触发）电活动的关键因素。因此，处理异位活动的心房Ca（2）处理异常可能构成治疗AF的新的基于机制的治疗方法。本文回顾了细胞异位活性在人类房颤病理生理中的作用的最新证据，讨论了在人类心房肌细胞中触发活动的潜在分子机制，并考虑了它们与新型治疗方案设计的相关性。

BACKGROUND & AIMS: :Synaptosomes isolated from adult rat cerebral cortices were used for studying the uptake of L-leucine by the Na(+)-dependent route. Three non-metabolizable amino acid analogues, which had been used previously to discriminate the Na(+)-dependent A-type uptake system of animal cells, were employed in this study. It was found that Na(+)-dependent uptake of leucine was insensitive to inhibition by 2-aminoisobutyric acid (AIB) and N-methylaminoisobutyric acid (MeAIB) whereas N-methylalanine (NMA) was markedly inhibitory. Inhibition by NMA was stereospecific--only the L-isomer had a pronounced effect. Na(+)-dependent uptake of leucine as well as its inhibition by L-NMA were rather insensitive to changes in pH from 6 to 9. Kinetic analysis of inhibition by L-NMA of Na(+)-dependent uptake revealed a non-competitive type of inhibition with a Ki value of approximately 0.5 mM.

背景与目标： ：从成年大鼠大脑皮层分离的突触体用于研究Na（）依赖途径对L-亮氨酸的摄取。在这项研究中使用了三种不可代谢的氨基酸类似物，这些氨基酸类似物先前已用于区分动物细胞的Na（-）依赖性A型摄取系统。发现亮氨酸的Na（）依赖性摄取对2-氨基异丁酸（AIB）和N-甲基氨基异丁酸（MeAIB）的抑制不敏感，而N-甲基丙氨酸（NMA）具有明显的抑制作用。 NMA的抑制作用是立体特异性的-只有L-异构体具有明显的作用。 Na（）依赖性亮氨酸的摄取及其对L-NMA的抑制作用对pH值从6到9的变化不敏感。动力学分析表明L-NMA对Na（）依赖性摄取的抑制作用揭示了一种非竞争性类型Ki值约为0.5 mM的抑制作用。

BACKGROUND & AIMS: The human immunodeficiency virus type 1 (HIV-1) Tat protein strongly transactivates gene expression from the viral long terminal repeat (LTR) and is required for virus efficient replication. Previous studies have shown that cells scrape-loaded in the presence of purified recombinant Tat can absorb the protein in a receptor-independent fashion. Using recombinant Tat in which cysteine residues were blocked by sulfitolysis to prevent disulfide aggregation (S-Tat) we were unable to observe this phenomenon, possibly because of improper protein folding. In this study we report that the block to cellular uptake could be overcome by mixing S-Tat with a cationic liposome, Lipofectin. When mixed with Lipofectin, S-Tat effected a specific, concentration-dependent transactivation of HIV-1 LTR-directed reporter gene activity in Hela Cells. Cellular uptake was confirmed by Western blot analysis with an anti-Tat antibody. The method described utilizes cells plated in a 96-well format, requires only nanogram quantities of S-Tat protein and is much less labor-intensive than assays involving scrape-loading, making it suitable for use as a high-throughput screen for detecting Tat inhibitors. The method may have applications for the analysis of other recombinant proteins that require uptake into intact cells for determination of functionality and presents a general technique for introducing exogenous proteins into cells.

背景与目标： 人类免疫缺陷病毒1型（HIV-1）Tat蛋白可以强烈地激活病毒长末端重复序列（LTR）的基因表达，并且是病毒有效复制所必需的。先前的研究表明，在纯化的重组Tat存在下，被刮擦的细胞可以以不依赖受体的方式吸收蛋白质。使用重组Tat，其中半胱氨酸残基被亚硫酸盐分解作用阻止，以防止二硫键聚集（S-Tat），我们无法观察到这种现象，这可能是由于蛋白质折叠不当所致。在这项研究中，我们报道通过将S-Tat与阳离子脂质体Lipofectin混合可以克服对细胞摄取的阻碍。与Lipofectin混合后，S-Tat在Hela细胞中实现了HIV-1 LTR指导的报告基因活性的特异性，浓度依赖性反式激活。用抗Tat抗体通过蛋白质印迹分析确认了细胞摄取。所描述的方法利用以96孔格式铺板的细胞，仅需纳克量的S-Tat蛋白，并且比涉及刮擦试验的劳动强度低得多，使其适合用作检测Tat的高通量筛选抑制剂。该方法可用于分析其他重组蛋白质，这些蛋白质需要摄取完整细胞来确定功能性，并提出了将外源蛋白质引入细胞的一般技术。

BACKGROUND & AIMS: :We tried to evaluate the possible involvement of fetuin in the scavenger receptors (SRs)-mediated hepatic uptake of polystyrene nanospheres with the size of 50 nm (NS-50), which has surface negative charge (zeta potential=-21.8+/-2.3 mV). The liver perfusion studies in rats revealed that the hepatic uptake of NS-50 pre-coated with fetuin (NS-50-fetuin) was significantly inhibited by poly inosinic acid (poly I), a typical inhibitor of SRs, whereas that of plain NS-50 or NS-50 pre-coated with BSA (NS-50-BSA) was not. The uptake of NS-50-fetuin by cultured Kupffer cells was also significantly inhibited by poly I, and anti-class A scavenger receptors (SR-A) antibody, suggesting that fetuin on NS-50 mediated the recognition and internalization of NS-50 by Kupffer cells and at least SR-A would be responsible for the uptake. Taken that Western blot analysis confirmed that fetuin certainly adsorbed on the surface of NS-50 after the incubation of NS-50 with serum, the results obtained in the present study indicate that fetuin would be one of the serum proteins that were substantially involved in the hepatic uptake of NS-50 via SRs.

背景与目标： ：我们试图评估胎球蛋白可能参与清道夫受体（SRs）介导的肝脏吸收大小为50 nm（NS-50）的聚苯乙烯纳米球，该表面具有表面负电荷（ζ电位= -21.8 /-2.3 mV）。在大鼠的肝脏灌注研究中发现，典型的SR抑制剂聚肌苷酸（poly I）显着抑制了预先涂有胎球蛋白（NS-50-胎球蛋白）的NS-50对肝的摄取。未预涂BSA的-50或NS-50（NS-50-BSA）。聚I和抗A类清道夫受体（SR-A）抗体也显着抑制了培养的Kupffer细胞对NS-50-胎球蛋白的摄取，表明NS-50上的胎球蛋白介导了NS-50的识别和内在化。枯否细胞的吸收，至少由SR-A引起。认为Western印迹分析证实胎球蛋白在将血清与NS-50孵育后肯定吸附在NS-50的表面上，本研究获得的结果表明胎球蛋白将是实质性参与胎盘蛋白的血清蛋白之一。肝通过SRs吸收NS-50。