BACKGROUND & AIMS: :We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

背景与目标： : 我们以前已经表明，Ca2/钙调蛋白与代谢型谷氨酸受体7 (mGluR7) 的相互作用促进了g蛋白介导的对激动剂激活后看到的电压敏感Ca2通道 (vscc) 的抑制。在这里，我们使用mGluR7的细胞质C末端尾巴作为诱饵进行了新生大鼠脑cDNA文库的酵母双杂交筛选，并确定了巨噬细胞肉豆蔻基化的富含丙氨酸的c激酶底物 (MacMARCKS) 作为结合蛋白。通过下拉测定，免疫沉淀以及mGluR7和MacMARCKS在转染的HEK293细胞和培养的小脑颗粒细胞中的共定位，在体外和体内证实了相互作用。Ca2 +/钙调蛋白拮抗MacMARCKS与mGluR7的结合。在神经元中，在没有mGluR7激动剂的情况下，MacMARCKS与mGluR7共转染，但不能与MacMARCKS结合的mGluR7突变体，降低了g蛋白介导的VSCCs的强直抑制。这些结果表明，Ca2/钙调蛋白和MacMARCKS与mGluR7的竞争性相互作用控制了g蛋白对VSCCs的强直抑制。

BACKGROUND & AIMS: :Sepsis results in a state of relative immunosuppression, rendering critically ill patients susceptible to secondary infections and increased mortality. Monocytes isolated from septic patients and experimental animals display a "deactivated" phenotype, characterized by impaired inflammatory and antimicrobial responses, including hyporesponsiveness to LPS. We investigated the role of the LPS/TLR4 axis and its inhibitor, IL-1 receptor-associated kinase-M (IRAK-M), in modulating the immunosuppression of sepsis using a murine model of peritonitis-induced sepsis followed by secondary challenge by intratracheal Pseudomonasaeruginosa. Septic mice demonstrated impaired alveolar macrophage function and increased mortality when challenged with intratracheal Pseudomonas as compared with nonseptic controls. TLR2 and TLR4 expression was unchanged in the lung following sepsis, whereas levels of IRAK-M were upregulated. Macrophages from IRAK-M-deficient septic mice produced higher levels of proinflammatory cytokines ex vivo and greater costimulatory molecule expression in vivo as compared with those of their WT counterparts. Following sepsis and secondary intrapulmonary bacterial challenge, IRAK-M(-/-) animals had higher survival rates and improved bacterial clearance from lung and blood compared with WT mice. In addition, increased pulmonary chemokine and inflammatory cytokine production was observed in IRAK-M(-/-) animals, leading to enhanced neutrophil recruitment to airspaces. Collectively, these findings indicate that IRAK-M mediates critical aspects of innate immunity that result in an immunocompromised state during sepsis.

背景与目标： 败血症导致相对免疫抑制状态，使重症患者易继发感染并增加死亡率。从败血症患者和实验动物中分离出的单核细胞显示出 “失活” 表型，其特征是炎症和抗菌反应受损，包括对LPS的低反应性。我们研究了LPS/TLR4轴及其抑制剂IL-1受体相关激酶M (IRAK-M) 在调节脓毒症免疫抑制中的作用，该模型使用腹膜炎诱导的脓毒症，然后通过气管内假单胞菌继发攻击的鼠模型。与非败血症对照组相比，败血症小鼠的肺泡巨噬细胞功能受损，死亡率增加。脓毒症后肺中TLR2和TLR4表达不变，而IRAK-M水平上调。与WT对应物相比，来自IRAK-M缺陷脓毒症小鼠的巨噬细胞在体外产生更高水平的促炎细胞因子，并在体内产生更大的共刺激分子表达。与WT小鼠相比，在败血症和继发性肺内细菌攻击后，IRAK-M(-/-) 动物具有更高的存活率，并且从肺和血液中清除细菌。此外，在IRAK-M(-/-) 动物中观察到肺趋化因子和炎性细胞因子的产生增加，导致中性粒细胞向空气空间的募集增强。总的来说，这些发现表明IRAK-M介导了先天免疫的关键方面，从而导致败血症期间的免疫功能低下状态。

BACKGROUND & AIMS: :Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.

背景与目标： 角膜成纤维细胞 (CFs) 介导的胶原收缩与角膜形状的维持有关。鉴于纤连蛋白在角膜基质损伤部位表达，我们研究了纤连蛋白对CF介导的胶原凝胶收缩的影响。在不存在或存在各种细胞外基质 (ECM) 成分的情况下，在I型胶原蛋白的三维凝胶中培养人CFs。通过测量凝胶直径的变化来评估CFs介导的胶原蛋白凝胶的收缩。通过荧光显微镜检查CFs中应力纤维和粘着斑的形成。通过免疫印迹分析检查CFs中桩蛋白，磷酸化桩蛋白，整联蛋白 α5，β1和 α2以及 α-平滑肌肌动蛋白的丰度。纤连蛋白以浓度和时间依赖性方式促进CF介导的胶原蛋白凝胶收缩。其他ECM蛋白或糖胺聚糖没有表现出这种作用。纤连蛋白还诱导细胞扩散，应力纤维的形成以及在三维胶原蛋白凝胶中培养的CFs中建立含有桩蛋白的粘着斑。此外，它增加了这些细胞中的桩蛋白，磷酸化的桩蛋白以及整合素 α5和 β1的量。然而，整合素 α2和 α-平滑肌肌动蛋白的表达不受纤连蛋白的影响。此外，肽GRGDSP (纤连蛋白受体的拮抗剂) 阻断了纤连蛋白对CF介导的胶原蛋白凝胶收缩的刺激作用。这些结果表明，纤连蛋白以取决于应力纤维和粘着斑的形成，桩蛋白的活化以及整联蛋白alpha5beta1的上调的方式促进CF介导的胶原蛋白凝胶收缩。因此，纤连蛋白可能有助于CFs在基质伤口愈合过程中维持角膜形状。

BACKGROUND & AIMS: Development of methodologies for gene transfer into the central nervous system (CNS) is important for fundamental research as well as clinical studies for gene therapy. Cationic liposomes (CL) are attractive vectors because of their safety and ease of use. However, to date only low rates of success have been reported. We succeeded in obtaining high transfection efficiencies into the newborn mouse brain in vivo by CL and a cytoplasmic gene expression system based on T7 RNA polymerase and T7 RNA polymerase- and the luciferase-gene with the T7 promoter sequence. This system showed an efficiency rate 2 orders of magnitude higher than the standard system, which used CL and luciferase genes with a Rous sarcoma virus promoter, pRSVL. In addition, in vitro experiments using LLCMK2 cells showed that cytoplasmic gene expression occurred rapidly (within 6 h) after transfection. In contrast, pRSVL required 24-48 h for induction of luciferase expression. Our results suggest that the cytoplasmic gene expression system is useful for gene delivery into the CNS.

背景与目标： 开发将基因转移到中枢神经系统 (CNS) 的方法对于基因治疗的基础研究和临床研究至关重要。阳离子脂质体 (CL) 是有吸引力的载体，因为它们的安全性和易用性。然而，迄今为止，只有低成功率的报道。我们成功地通过CL和基于T7 RNA聚合酶和T7 RNA聚合酶以及具有T7启动子序列的荧光素酶基因的细胞质基因表达系统在体内获得了高转染效率。该系统的效率比标准系统高2个数量级，标准系统使用具有Rous肉瘤病毒启动子pRSVL的CL和荧光素酶基因。此外，使用LLCMK2细胞的体外实验表明，转染后细胞质基因表达迅速 (在6小时内) 发生。相反，pRSVL需要24-48小时才能诱导荧光素酶表达。我们的结果表明，细胞质基因表达系统可用于将基因传递到CNS中。

BACKGROUND & AIMS: In the initial stages of crystallization of proteins, monomers aggregate rapidly and form nuclei and large fractal clusters, as previously shown by dynamic light scattering experiments (Georgalis, Y., J. Schüler, J. Frank, D. M. Soumpasis, and W. Saenger. 1995. Protein crystallization screening through scattering techniques. Adv. Colloid Interface Sci. 5857-86). In this communication we initiate an effort to understand the effective interactions controlling charged protein aggregation and crystallization using the potential of mean force (PMF) theory. We compute the PMFs of the system lysozyme-water-NaCl within the framework of the hypernetted chain approximation for a wide range of protein and salt concentrations. We show that the computed effective interactions can rationalize the experimentally observed aggregation behavior of lysozyme under crystallization conditions.

背景与目标： 在蛋白质结晶的初始阶段，单体迅速聚集并形成核和大的分形簇，如先前的动态光散射实验所示 (Georgalis，Y.，J. Sch ü ler，J. Frank，d.m.Sompasis，和W. Saenger。1995。通过散射技术进行蛋白质结晶筛选。胶体界面科学。5857-86)。在此交流中，我们开始努力使用平均力 (PMF) 理论来了解控制带电蛋白质聚集和结晶的有效相互作用。我们在各种蛋白质和盐浓度的超净链近似框架内计算系统溶菌酶-水-NaCl的pmf。我们证明，计算出的有效相互作用可以使结晶条件下实验观察到的溶菌酶的聚集行为合理化。

BACKGROUND & AIMS: :Proteins are required for the efflux of heme from mitochondria and liposomes. The efflux from liposomes is independent of the heme-binding affinity of the protein (Biochem. 23:3715, 1984). We tested whether heme-binding proteins increase efflux of newly synthesized heme from structurally and functionally intact rat liver mitochondria. Mitochondria whose heme was labeled with 14C-delta-aminolevulinic acid, were incubated in the presence of glutathione transferases (GSTs), serum albumin (RSA) or heme-binding protein (HBP), all from the rat. HBP caused a 6-8 fold increase in efflux of newly synthesized heme as compared to that effected by RSA or GSTs. This result indicates that heme efflux from intact mitochondria, unlike that from liposomes, depends on the type of protein present and that HBP may specifically facilitate heme efflux from mitochondria.

背景与目标： : 线粒体和脂质体中血红素的流出需要蛋白质。来自脂质体的流出与蛋白质的血红素结合亲和力无关 (biochem23: 3715，1984)。我们测试了血红素结合蛋白是否增加了结构和功能完整的大鼠肝线粒体中新合成的血红素的流出。将血红素用14c-delta-氨基乙酰丙酸标记的线粒体在谷胱甘肽转移酶 (gst)，血清白蛋白 (RSA) 或血红素结合蛋白 (HBP) 的存在下孵育。与RSA或GSTs相比，HBP导致新合成血红素的流出增加了6-8倍。该结果表明，与脂质体不同，完整线粒体中的血红素流出取决于存在的蛋白质类型，并且HBP可能特别促进线粒体中的血红素流出。

BACKGROUND & AIMS: :Mouse hepatitis virus (MHV) is one of the most prevalent viruses detected in laboratory mouse colonies. Enterotropic strains predominate in natural infections, and molecular techniques for the detection of MHV shedding in feces are powerful enough to diagnose active infections. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technique was developed for the detection of rodent coronaviruses within 90 min. The specificity of this technique was confirmed by its ability to detect all 17 different strains of MHV and 6 strains of rat coronaviruses as well as its failure to detect human, bovine, and porcine coronaviruses nonspecifically. The sensitivity of RT-LAMP was 3.2-fold higher than that of reverse transcription-polymerase chain reaction (RT-PCR) and 31.6-fold lower than that of nested RT-PCR. An evaluation of the diagnostic performance of RT-LAMP performed in duplicate using mouse fecal specimens showed that the sensitivity and specificity with respect to nested RT-PCR were 85.7% and 100%, respectively. RT-LAMP assays would be suitable for monitoring active MHV infection in mouse colonies.

背景与目标： : 小鼠肝炎病毒 (MHV) 是在实验室小鼠菌落中检测到的最普遍的病毒之一。肠溶性菌株在自然感染中占主导地位，用于检测粪便中MHV脱落的分子技术足以诊断活动性感染。开发了一种逆转录环介导的等温扩增 (rt-lamp) 技术，用于在90分钟内检测啮齿动物冠状病毒。该技术的特异性通过其检测所有17种不同的MHV菌株和6种大鼠冠状病毒菌株的能力以及未能非特异性检测人，牛和猪冠状病毒的能力得到证实。Rt-lamp的敏感性比逆转录聚合酶链反应 (rt-pcr) 高3.2倍，比巢式rt-pcr低31.6倍。对使用小鼠粪便标本进行的rt-lamp的诊断性能的评估显示，相对于巢式rt-pcr的敏感性和特异性分别为85.7% 和100%。RT-LAMP检测将适用于监测小鼠菌落中活跃的MHV感染。

BACKGROUND & AIMS: :Triple-negative breast cancer is characterized by aggressive tumours whose cells lack oestrogen and progesterone receptors and do not over-express HER2. It accounts for approximately 10-15% of breast cancer cases. We sought to generate a cellular model of chemotherapy drug resistance for this type of disease to provide the tools for the development of new therapies. Doxorubicin is a component of some chemotherapy regimes used to treat this form of cancer but resistance preventing disease eradication frequently occurs, mainly due to over-expression of drug transporters such as P-glycoprotein. CALDOX cells were generated by exposure of CAL51 to doxorubicin. Resistance to doxorubicin did not involve drug transporters, as the both parental and resistant cells accumulated doxorubicin to comparable levels. CALDOX cells had slower proliferation rate and an extended G1 cell cycle stage than the parental line, mainly due to an intrinsic activation of CDNK1 (p21), but this cell cycle block was not involved in the mechanism of resistance. CALDOX cells had reduced levels of TOP2A (topoisomerase IIα) and were cross resistant to the topoisomerase II inhibitors etoposide and mitoxantrone. CALDOX cells showed collateral sensitivity to carmustine due to the lack of O⁶-methylguanine-DNA-methyltransferase (MGMT) expression, related to the hypermethylation of its promoter. The collateral sensitivity of CALDOX cells to carmustine provides the rationale to evaluate MGMT promoter methylation status to design better therapeutic strategies for triple negative breast cancer.

背景与目标： : 三阴性乳腺癌的特征是侵袭性肿瘤，其细胞缺乏雌激素和孕激素受体，并且不会过度表达her2。它约占乳腺癌病例的10-15%。我们试图为这种类型的疾病生成化学疗法耐药性的细胞模型，以为开发新疗法提供工具。阿霉素是用于治疗这种癌症的某些化学疗法方案的组成部分，但经常发生抗药性预防疾病根除，这主要是由于药物转运蛋白 (例如P-糖蛋白) 的过表达。CALDOX细胞是通过将CAL51暴露于阿霉素而产生的。对阿霉素的耐药性不涉及药物转运蛋白，因为亲本和耐药细胞都积累了阿霉素的水平相当。与亲本系相比，钙氧化细胞的增殖速度较慢，G1细胞周期阶段延长，这主要是由于CDNK1 (p21) 的内在激活，但这种细胞周期阻滞不参与抗性机制。钙氧化细胞的TOP2A (拓扑异构酶II α) 水平降低，并且对拓扑异构酶II抑制剂依托泊苷和米托蒽醌具有交叉抗性。由于缺乏o-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 表达，CALDOX细胞对卡莫司汀表现出附带敏感性，这与其启动子的高甲基化有关。CALDOX细胞对卡莫司汀的附带敏感性为评估MGMT启动子甲基化状态以设计更好的三阴性乳腺癌治疗策略提供了依据。

BACKGROUND & AIMS: :The objective of this study was to evaluate the effects of ultrasound-mediated analyte diffusion on permeability of normal, benign, and cancerous human lung tissue in vitro and to find more effective sonophoretic (SP) delivery in combination with the optical clearing agents (OCAs) method to distinguish normal and diseased lung tissues. The permeability coefficients of SP in combination with OCAs diffusion in lung tissue were measured with Fourier-domain optical coherence tomography (FD-OCT). 30% glucose and SP with a frequency of 1 MHz and an intensity of 0.80  W/cm2 over a 3 cm probe was simultaneously applied for 15 min. Experimental results show that the mean permeability coefficients of 30% glucose/SP were found to be (2.01±0.21)×10(-5)  cm/s from normal lung (NL) tissue, (2.75±0.28)×10(-5)  cm/s from lung benign granulomatosis (LBG) tissue, (4.53±0.49)×10(-5)  cm/s from lung adenocarcinoma tumor (LAT) tissue, and (5.81±0.62)×10(-5)  cm/s from lung squamous cell carcinoma (LSCC) tissue, respectively. The permeability coefficients of 30% glucose/SP increase approximately 36.8%, 125.4%, and 189.1% for the LBG, LAT, and LSCC tissue compared with that for the NL tissue, respectively. There were statistically significant differences in permeability coefficients of 30% glucose/SP between LBG and NL tissue (p<0.05), between LAT and NL tissue (p<0.05), and between LSCC and NL tissue (p<0.05). The results suggest that the OCT functional imaging technique to combine an ultrasound-OCAs combination method could become a powerful tool in early diagnosis and monitoring of changed microstructure of pathologic human lung tissue.

背景与目标： : 这项研究的目的是评估超声介导的分析物扩散对体外正常，良性和癌性人肺组织通透性的影响，并找到与光学清除剂 (OCAs) 结合更有效的超声电泳 (SP) 递送方法，以区分正常和患病的肺组织。用傅里叶域光学相干断层扫描 (fd-oct) 测量SP与OCAs扩散在肺组织中的渗透系数。在3厘米探针上同时施加频率为1 MHz且强度为0.80   W w/cm2的30% 葡萄糖和SP 15分钟。实验结果表明，30% 葡萄糖/SP对正常肺 (NL) 组织的平均渗透系数为 (2.01 ± 0.21)× 10(-5)  cm cm/s，肺良性肉芽肿 (LBG) 组织 (2.75 ± 0.28)× 10(-5)  cm/s，肺腺癌肿瘤 (LAT) 组织 (4.53 ± 0.49)× 10(-5)  cm/s，和 (5.81 ± 0.62)× 10(-5)  cm cm/s分别来自肺鳞状细胞癌 (LSCC) 组织。与NL组织相比，LBG、LAT和LSCC组织的30% 葡萄糖/SP的渗透系数分别增加约36.8% 、125.4% 和189.1%。LBG与NL组织之间 (p<0.05)，LAT与NL组织之间 (p<0.05) 以及LSCC与NL组织之间 (p<0.05) 的30% 葡萄糖/SP的渗透系数差异有统计学意义。结果表明，OCT功能成像技术结合超声-OCAs组合方法可以成为早期诊断和监测病理性人肺组织微观结构变化的有力工具。

BACKGROUND & AIMS: :Bile acids are synthesized in the liver and are the major component in bile. Impaired bile flow leads to cholestasis that is characterized by elevated levels of bile acid in the liver and serum, followed by hepatocyte and biliary injury. Although the causes of cholestasis have been extensively studied, the molecular mechanisms as to how bile acids initiate liver injury remain controversial. In this chapter, we summarize recent advances in the pathogenesis of bile acid induced liver injury. These include bile acid signaling pathways in hepatocytes as well as the response of cholangiocytes and innate immune cells in the liver in both patients with cholestasis and cholestatic animal models. We focus on how bile acids trigger the production of molecular mediators of neutrophil recruitment and the role of the inflammatory response in this pathological process. These advances point to a number of novel targets where drugs might be judged to be effective therapies for cholestatic liver injury.

背景与目标： : 胆汁酸在肝脏中合成，是胆汁中的主要成分。胆汁流动受损导致胆汁淤积，其特征是肝脏和血清中胆汁酸水平升高，随后是肝细胞和胆道损伤。尽管胆汁淤积的原因已得到广泛研究，但有关胆汁酸如何引发肝损伤的分子机制仍存在争议。在本章中，我们总结了胆汁酸引起的肝损伤的发病机理的最新进展。这些包括胆汁淤积和胆汁淤积动物模型患者的肝细胞中的胆汁酸信号通路以及肝脏中胆管细胞和先天免疫细胞的反应。我们专注于胆汁酸如何触发中性粒细胞募集分子介质的产生以及炎症反应在此病理过程中的作用。这些进展指出了许多新的靶点，在这些靶点中，药物可能被认为是治疗胆汁淤积性肝损伤的有效疗法。

BACKGROUND & AIMS: Background:How plants adapt their developmental patterns to regular seasonal changes is an important question in biology. The annual growth cycle in perennial long-lived trees is yet another example of how plants can adapt to seasonal changes. The two main signals that plants rely on to respond to seasonal changes are photoperiod and temperature, and these signals have critical roles in the temporal regulation of the annual growth cycle of trees. Scope:This review presents the latest findings to provide insight into the molecular mechanisms that underlie how photoperiodic and temperature signals regulate seasonal growth in trees. Conclusion:The results point to a high level of conservation in the signalling pathways that mediate photoperiodic control of seasonal growth in trees and flowering in annual plants such as arabidopsis. Furthermore, the data indicate that symplastic communication may mediate certain aspects of seasonal growth. Although considerable insight into the control of phenology in model plants such as poplar and spruce has been obtained, the future challenge is extending these studies to other, non-model trees.

背景与目标：

BACKGROUND & AIMS: OBJECTIVES:To evaluate in vitro and in vivo a strategy for gene therapy for AIDS based on the transfer on interferon (IFN)-alpha, -beta and -gamma genes to human cells.

DESIGN:Human U937 promonocytic cells were stably transfected with Tat-inducible IFN expression vectors conferring an antiviral state against infection with HIV.

METHODS:Transfected cells were either infected by HIV-1 in vitro or transplanted into severe combined immunodeficient (SCID) mice for an HIV challenge in vivo.

RESULTS:U937 cell lines stably carrying IFN transgenes under the positive control of the HIV-1 Tat protein were highly resistant to HIV-1 replication in vitro. This antiviral resistance was associated with a strong induction of IFN synthesis immediately following the viral infection. HIV-1 proteins were found to be specifically trapped within the genetically modified cells. In contrast, all IFN-U937 cells permitted full HIV-2 replication. Transfected cells injected into SCID mice and challenged against HIV-1 were strongly resistant to infection when cells were transduced with IFN-alpha of IFN-beta genes. However, IFN-gamma-transfected cells permitted HIV-1 infection in vivo despite the induction of a high level of IFN-gamma secretion. The quantity of proviral DNA was 10(5)-fold lower in IFN-alpha- or IFN-beta-transfected U937 cells collected from these SCID mice than that in non-transfected cells.

CONCLUSIONS:Our results substantiated the validity of a strategy, bases on the transfer of HIV-1-inducible IFN-alpha or IFN-beta genes, to confer antiviral resistance to human cells.

背景与目标： 目标 : 基于干扰素 (IFN)-α，-β 和-γ 基因向人类细胞的转移，在体外和体内评估用于艾滋病的基因治疗策略。
设计 : 人U937单核细胞用Tat诱导的IFN表达载体稳定转染，赋予抗HIV感染的抗病毒状态。
方法 : 转染的细胞要么通过体外HIV-1感染，要么移植到严重的联合免疫缺陷 (SCID) 小鼠体内进行HIV攻击。
结果 : 在HIV-1 Tat蛋白的阳性控制下稳定携带IFN转基因的U937细胞系在体外对HIV-1复制具有高度抗性。这种抗病毒耐药性与病毒感染后立即强烈诱导IFN合成有关。发现HIV-1蛋白质被特异性地捕获在转基因细胞内。相反，所有IFN-U937细胞允许完全HIV-2复制。当用IFN-β 基因的IFN-α 转导细胞时，注入SCID小鼠并对HIV-1进行攻击的转染细胞对感染具有强烈抗性。然而，尽管诱导了高水平的IFN-γ 分泌，但转染IFN-γ 的细胞允许体内HIV-1感染。从这些SCID小鼠收集的IFN-α 或IFN-β 转染的U937细胞中前病毒DNA的数量比未转染的细胞低10(5) 倍。
结论 : 我们的结果证实了基于HIV-1-inducible IFN-α 或IFN-β 基因转移的策略对人类细胞具有抗病毒抗性的有效性。

BACKGROUND & AIMS: :The human immunodeficiency virus type-1 (HIV-1) envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. Here, we show that N(CCG)-gp41, a class 2 inhibitor, and N36(Mut(e,g)), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudo-typed virus neutralization assay. The mechanistic, as well as potential therapeutic, implications of these observations for HIV-Env-mediated membrane fusion are discussed.

背景与目标： : 介导膜融合的人类免疫缺陷病毒1型 (HIV-1) 包膜 (Env) 蛋白是开发新的AIDS疗法的主要目标。已经描述了三类Env介导的膜融合抑制剂，它们专门针对gp41的发夹前中间构象。2类抑制剂与gp41的C端七药重复 (c-hr) 结合。3类抑制剂的单个实例针对gp41的三聚体N末端七位重复序列 (n-hr)，并已被假定通过形成融合无能的异三聚体来隔离发夹前中间体的n-hr。在这里，我们显示了2类抑制剂N(CCG)-gp41和3类抑制剂N36(Mut(e，g))，在细胞融合试验 (具有膜结合的CD4) 和Env-伪型病毒中和试验中，协同抑制几种代表性HIV-1菌株 (X4和R5) 的Env介导的膜融合。讨论了这些观察结果对HIV-Env介导的膜融合的机理以及潜在的治疗意义。

BACKGROUND & AIMS: We have recently shown that the incubation of Xenopus laevis oocytes in procaine-containing solutions induced germinal vesicle breakdown without white spot formation and, in some cases, with the appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine whether M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine, which are both known to inhibit p34cdc2 kinase. Histone H1 kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls. A shift in p34cdc2 was observed in oocytes that had been exposed to procaine for 16 h, but it was not detected in those exposed for 24 h. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occurred in oocytes incubated in procaine for 16 h could induce maturation of recipient stage VI oocytes. This transferable activity was weaker than that from progesterone-treated controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed, which were not always correctly located. Taken together these results demonstrate that M-phase promoting factor is involved in the procaine maturing effect despite some differences compared with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate M-phase promoting factor might now help in the understanding of some steps in progesterone-induced maturation that have still to be elucidated.

背景与目标： 我们最近表明，在含普鲁卡因的溶液中孵育非洲爪蟾卵母细胞可诱导生发囊泡破裂，而不会形成白斑，在某些情况下，细胞质中会出现纺锤体和染色体。进行本研究是为了确定M期促进因子是否参与了这种异常成熟。普鲁卡因在存在6-二甲基氨基嘌呤或roscovitine的情况下未能诱导成熟，这两者均已知会抑制p34cdc2激酶。在普鲁卡因处理的卵母细胞中检测到组蛋白H1激酶活性，但始终低于孕酮处理的对照组。在暴露于普鲁卡因16小时的卵母细胞中观察到p34cdc2的变化，但在暴露24小时的卵母细胞中未检测到p34cdc2的变化。最后，细胞质转移实验表明，在普鲁卡因中孵育16小时的卵母细胞中发生的促进成熟的活性可以诱导受体VI期卵母细胞的成熟。这种可转移活性比黄体酮治疗的对照组更弱，因为只有30% 的接受者经历了生发囊泡破裂，并且仅观察到几个纺锤体，这些纺锤体并不总是正确定位。综上所述，这些结果表明，尽管与孕酮处理的卵母细胞相比存在一些差异，但M期促进因子参与了普鲁卡因的成熟作用，这可能解释了该物质诱导的特定成熟类型。普鲁卡因能够激活M期促进因子的机制的发现现在可能有助于理解孕酮诱导的成熟中的一些步骤，这些步骤仍有待阐明。

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蛋白强烈反式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，在存在纯化的重组Tat的情况下，刮擦加载的细胞可以以不依赖于受体的方式吸收蛋白质。使用重组Tat，其中半胱氨酸残基被磺化酶分解阻断以防止二硫键聚集 (S-Tat)，我们无法观察到这种现象，可能是由于蛋白质折叠不当。在这项研究中，我们报告说，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滞。当与Lipofectin混合时，S-Tat在Hela细胞中实现了HIV-1 LTR导向的报告基因活性的特异性，浓度依赖性反式激活。通过抗Tat抗体的Western印迹分析证实了细胞摄取。所描述的方法利用了以96孔形式铺板的细胞，仅需要纳克量的S-Tat蛋白，并且比涉及刮擦加载的测定的劳动强度低得多，因此适合用作检测Tat的高通量筛选抑制剂。该方法可用于分析需要吸收到完整细胞中以确定功能的其他重组蛋白，并提出了将外源蛋白引入细胞的通用技术。