BACKGROUND & AIMS: :Under normoxic conditions the alpha-subunit of hypoxia-inducible factor (HIF-1alpha) protein is targeted for degradation by the von Hippel-Lindau (VHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Recently, we developed a hypoxia-targeting protein, TOP3, which consisted of procaspase-3 with the VHL-mediated protein destruction motif of HIF-1alpha. This design enables procaspase-3 to be regulated similarly with HIF-1alpha, being degraded under normoxia while stabilized under hypoxia. Furthermore, stabilized TOP3 was cleaved by the hypoxic stress-induced endogenous caspases and thus the procaspase-3 was converted to active caspase-3 specifically under hypoxic conditions. These data demonstrated that the VHL-mediated protein destruction motif of HIF-1alpha endowed procaspase-3 with hypoxia-specific cytotoxicity.

背景与目标： : 在常氧条件下，低氧诱导因子 (HIF-1alpha) 蛋白的 α 亚基被作为E3泛素连接酶的von Hippel-Lindau (VHL) 肿瘤抑制蛋白靶向降解。最近，我们开发了一种低氧靶向蛋白TOP3，它由具有HIF-1alpha VHL介导的蛋白破坏基序的procaspase-3组成。该设计使procaspase-3能够与HIF-1alpha类似地调节，在常氧条件下降解，而在缺氧条件下稳定。此外，稳定的TOP3被低氧胁迫诱导的内源性半胱天冬酶裂解，因此procaspase-3在低氧条件下被特异性转化为活性caspase-3。这些数据表明，HIF-1alpha的VHL介导的蛋白质破坏基序赋予procaspase-3低氧特异性细胞毒性。

BACKGROUND & AIMS: :Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

背景与目标： : 富含胶原蛋白的组织中的基质组装和稳态由与被硫酸化糖胺聚糖 (gag) 取代的蛋白聚糖 (pg) 的相互作用介导。角膜中的主要GAG是硫酸角质素 (KS)，它与三种PG核心蛋白之一N连接。为了确定KS功能中碳水化合物链硫酸化步骤的重要性，我们产生了在Chst5中具有靶向基因缺失的小鼠品系，该品系编码与KS链硫酸化不可或缺的N-acetylglucosamine-6-O-sulfotransferase。纯合突变体的角膜比WT或杂合小鼠的角膜明显薄。它们缺乏高硫酸化的KS，但含有主要角膜KSPG的核心蛋白lumican。组织化学染色的kspg与WT角膜中的原纤维胶原共相关，但在Chst5-null组织中未鉴定。相反，异常大的硫酸软骨素/硫酸皮肤素PG复合物在整个Chst5-deficient角膜中都很丰富，表明突变角膜中控制的PG产生发生了变化。Chst5-null小鼠的角膜基质在胶原原纤维结构中表现出广泛的结构改变，包括减少的纤维间间距和更多的空间混乱的胶原阵列。因此，KS GAG链的酶促硫酸化被确定为PG生物合成和胶原基质组织的关键要求。

BACKGROUND & AIMS: :How neutrophils (polymorphonuclear neutrophils, PMNs) damage vessels in leukocytoclastic vasculitis (LcV) mediated by immune complexes (ICs) is unclear. If degradative enzymes and oxygen radicals are released from PMNs while adhering to the inner side of the vessel wall, they could be washed away by the blood stream or neutralized by serum protease inhibitors. We investigated if in LcV PMNs could damage vessels from the tissue side after transmigration. We used CD18-deficient (CD18-/-) mice because the absence of CD18 excludes transmigration of PMNs. When eliciting the Arthus reaction in ears of CD18-/- mice, deposition of ICs was not sufficient to recruit PMNs or to induce IC-mediated LcV. Injection of PMNs intradermally in CD18-/- mice allowed us to investigate if bypassing diapedesis and placing PMNs exclusively on the abluminal side leads to vascular destruction. We found that injected PMNs gathered around perivascular ICs, but did not cause vessel damage. Only intravenous injection of wild-type PMNs could re-establish the Arthus reaction in CD18-/- mice. Thus, PMNs cause vessel damage during diapedesis from the luminal side, but not from the perivascular space. We suggest that in order to shield the cytotoxic products from the blood stream, ICs induce particularly tight interactions between them, PMNs and endothelial cells.

背景与目标： : 中性粒细胞 (多形核中性粒细胞，PMNs) 如何损害由免疫复合物 (ICs) 介导的白细胞碎裂血管炎 (LcV) 中的血管尚不清楚。如果降解酶和氧自由基从pmn中释放出来，同时粘附在血管壁的内侧，则它们可能会被血流冲走或被血清蛋白酶抑制剂中和。我们调查了在LcV中，pmn是否会在迁移后从组织侧损坏血管。我们使用CD18-deficient (CD18-/-) 小鼠，因为缺少CD18排除了pmn的迁移。当在CD18-/-小鼠的耳朵中引起Arthus反应时，ICs的沉积不足以募集pmn或诱导IC介导的LcV。在CD18-/-小鼠中皮内注射pmn使我们能够研究是否绕过血管造影并仅将pmn放置在管腔侧会导致血管破坏。我们发现注射的pmn聚集在血管周围ICs周围，但不会引起血管损伤。只有静脉注射野生型PMNs才能在CD18-/-小鼠中重新建立Arthus反应。因此，pmn在从管腔侧 (而不是从血管周围空间) 进行的排尿过程中会引起血管损伤。我们建议，为了从血流中屏蔽细胞毒性产物，ICs在它们，pmn和内皮细胞之间诱导特别紧密的相互作用。

BACKGROUND & AIMS: :The mechanism by which T cells signal other T cells is not well defined. This was investigated by studying the ability of circulating T cells to induce the proliferation of autologous T cell clones. Peripheral blood T cells activated by cross-linking of the CD3/T cell receptor complex, which increased the expression of cell adhesion molecules LFA-1, LFA-3 and ICAM-1, induced the proliferation of autologous T cell clones. Irradiated antigen-activated peripheral blood T cells could also induce the proliferation of T cell clones which could not recognize that antigen. T-T cell activation required cell contact, was not major histocompatibility complex (MHC) restricted and was blocked by monoclonal antibodies directed against adhesion molecules CD2 and LFA-3 but was not blocked by antibody to class II MHC determinants. As CD2 is the natural ligand for LFA-3, increased expression of T cell surface adhesion molecules LFA-1, ICAM-1 and particularly LFA-3 during an inflammatory response may rapidly recruit T cells that are activated through the CD2 pathway. These results allow a simplified model to explain how relatively few antigen/MHC-specific T cells can recruit large numbers of non-antigen-specific T cells in the generation of an inflammatory response and postulates a novel role of the CD2 molecule in T cell immune function.

背景与目标： : T细胞向其他T细胞发出信号的机制尚不明确。通过研究循环T细胞诱导自体T细胞克隆增殖的能力进行了研究。通过CD3/T细胞受体复合物交联激活的外周血T细胞，增加细胞粘附分子LFA-1、LFA-3和ICAM-1的表达，诱导自体T细胞克隆增殖。辐照的抗原激活的外周血T细胞也可以诱导无法识别该抗原的T细胞克隆的增殖。T-t细胞活化需要细胞接触，不受主要组织相容性复合物 (MHC) 的限制，并被针对粘附分子CD2和LFA-3的单克隆抗体阻断，但未被针对II类MHC决定簇的抗体阻断。由于CD2是LFA-3的天然配体，因此在炎症反应期间LFA-1、ICAM-1并且特别是LFA-3的T细胞表面粘附分子的表达增加可以快速募集通过CD2途径激活的T细胞。这些结果允许一个简化的模型来解释相对较少的抗原/MHC特异性T细胞如何在炎症反应的产生中募集大量非抗原特异性T细胞，并假设CD2分子在T细胞免疫功能中的新作用。

BACKGROUND & AIMS: :Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell.

背景与目标： : 关于人类免疫缺陷病毒1型 (HIV-1) 基因组RNA如何在细胞质中运输的细节知之甚少。该过程的一部分受异质核核糖核蛋白A2 (hnRNP A2) 的活性控制。本研究研究了hnRNP A2在HeLa细胞中真正的前病毒表达中的作用。使用免疫荧光和荧光原位杂交技术，我们显示了HIV-1-expressing细胞中hnRNP A2表达的敲低导致HIV-1基因组RNA在与微管组织中心 (MTOC) 相对应的独特细胞质空间中快速积累。即使在hnRNP A2-response元件 (A2RE) 中携带突变的前病毒的表达过程中，由于hnRNP A2敲除，RNA也会在细胞核中离开并在MTOC区域积累，其表达导致基因组RNA的核保留。我们还证明hnRNP A2表达是细胞质中来自MTOC的基因组RNA下游运输所必需的。hnRNP A2敲低的结果和Rab7-interacting溶酶体蛋白的过表达在MTOC上的基因组RNA定位对pr55Gag的合成几乎没有影响，但对病毒的产生和感染性产生负面影响。这些数据表明，改变的HIV-1基因组RNA定位调节病毒组装，并且MTOC充当HIV-1基因组RNA从细胞核退出后会聚的中心位点，宿主蛋白hnrnpa2在将其带到和从细胞中的该位点中起着核心作用。

BACKGROUND & AIMS: BACKGROUND AND AIMS OF THE STUDY:Bovine and porcine pericardial tissues stabilized by dye-mediated photooxidation have found application as bioprosthetic heart valve material.

METHODS:To help predict clinical performance, a series of tests were performed to assess the biocompatibility and immunologic properties of these materials.

RESULTS AND CONCLUSIONS:Photooxidized bovine or porcine pericardium sterilized with an iodine-based solution were found to be non-cytotoxic, non-hemolytic, and non-mutagenic. Oil or saline extracts of these tissues passed tests for intracutaneous toxicity (irritation), acute systemic toxicity, and subchronic toxicity. Histopathology of 90-day implants of these tissues in the rabbit model demonstrated no significant macroscopic reaction and only slight microscopic response. Using a rabbit model to assess immune response, both bovine and porcine pericardial tissues elicited low levels of antibody. Furthermore, tissue photooxidation or iodine sterilization did not increase the overall level of antibodies. Glutaraldehyde-treated tissue also elicited low antibody levels which were higher than photooxidized tissue-induced levels. Absorption studies indicated that the photooxidation process may generate new epitopes, possibly collagen cross-links. Using the juvenile sheep model to assess in vivo performance, bioprosthetic valves made with photooxidized tissue were implanted and allowed to serve as functional implants for up to two years. Upon explant, the photooxidized pericardial leaflets were found to be non-calcific and partially covered with a layer of host cells. Histological cross-sections stained with von Willebrand's factor confirmed this layer as endothelial cells.

背景与目标： 研究的背景和目的 : 通过染料介导的光氧化作用稳定的牛和猪心包组织已被用作生物人工心脏瓣膜材料。
方法 : 为了帮助预测临床表现，进行了一系列测试以评估这些材料的生物相容性和免疫学特性。
结果和结论 : 发现用碘基溶液灭菌的光氧化牛或猪心包具有非细胞毒性，非溶血性，和非诱变性。这些组织的油或盐水提取物通过了皮内毒性 (刺激)，急性全身毒性和亚慢性毒性的测试。在兔模型中，这些组织的90天植入物的组织病理学没有明显的宏观反应，只有轻微的微观反应。使用兔模型评估免疫反应，牛和猪心包组织均引起低水平的抗体。此外，组织光氧化或碘灭菌不会增加抗体的总体水平。戊二醛处理的组织也引起低抗体水平，该水平高于光氧化组织诱导的水平。吸收研究表明，光氧化过程可能会产生新的表位，可能是胶原蛋白交联。使用幼年绵羊模型评估体内性能，将用光氧化组织制成的生物人工瓣膜植入并作为功能性植入物长达两年。外植体后，发现光氧化的心包小叶是非钙化的，并部分覆盖有一层宿主细胞。用von Willebrand因子染色的组织学横截面证实了该层为内皮细胞。

BACKGROUND & AIMS: :A cDNA encoding a novel human extracellularly-regulated kinase (ERK) phosphatase, designated B59, was isolated from a B5/589 human mammary epithelial cell cDNA library. The 1104 nucleotide open reading frame encodes 368 amino acids including the highly conserved catalytic site sequence of protein phosphotyrosine phosphatases (PTPs), VXVHCXXGXXR, at amino acid position 276-287. The predicted 70 amino acid stretch surrounding the HC motif shares significant sequence identity with other human dual specificity PTPs (dsPTPs), including the known ERK PTPs CL100, PAC1, B23, as well as the dsPTPs VH-1 and VHR. B59 protein synthesized in vitro in a rabbit reticulocyte lysate dephosphorylated rat ERK1 and ERK2 proteins whose phosphorylation had been stimulated by v-mos kinase added to the lysate. Ectopic expression of B59 in NIH3T3 fibroblasts inhibited the induction of an oncogene-responsive promoter by the dominant-activating raf mutant, raf-BXB. Moreover, cotransfection of NIH3T3 cells with B59 inhibited morphological transformation by H-ras and v-raf oncogenes. These results suggest that B59 suppresses the transforming activity of H-ras or v-raf oncogenes through ERK dephosphorylation and inactivation.

背景与目标： : 从B5/589人乳腺上皮细胞cDNA文库中分离编码新的人细胞外调节激酶 (ERK) 磷酸酶的cDNA，命名为B59。1104核苷酸开放阅读框编码368个氨基酸，包括蛋白质磷酸酪氨酸磷酸酶 (ptp) 的高度保守的催化位点序列VXVHCXXGXXR，在氨基酸位置276-287。HC基序周围的预测的70个氨基酸延伸与其他人类双特异性PTPs (dsPTPs) 具有显着的序列同一性，包括已知的ERK PTPs CL100，PAC1，B23以及dsPTPs VH-1和VHR。在兔网织红细胞裂解物中体外合成的B59蛋白去磷酸化的大鼠ERK1和ERK2蛋白，其磷酸化已被添加到裂解物中的v-mos激酶刺激。NIH3T3成纤维细胞中B59的异位表达抑制了显性激活raf突变体raf-BXB诱导癌基因应答启动子。此外，用B59共转染NIH3T3细胞可抑制H-ras和v-raf癌基因的形态转化。这些结果表明，B59通过ERK去磷酸化和失活抑制H-ras或v-raf癌基因的转化活性。

BACKGROUND & AIMS: :Endomyocardial specimens were obtained from 7 patients with acute myocarditis. Immunohistochemical examination of the mononuclear infiltrate showed mainly cytotoxic T lymphocytes and natural killer cells. Perforin (a pore-forming protein found in cytotoxic lymphocytes) was identified in this myocardial lymphocytic infiltrate and electron microscopy showed myocardial cell damage that may have been associated with these perforin containing lymphocytes. The results indicate that in acute idiopathic and viral myocarditis, myocardial damage may be due to the action of perforin-secreting lymphocytes.

背景与目标： : 从7例急性心肌炎患者中获得心内膜心肌标本。对单核浸润的免疫组织化学检查显示主要是细胞毒性T淋巴细胞和自然杀伤细胞。在这种心肌淋巴细胞浸润中鉴定出穿孔素 (一种在细胞毒性淋巴细胞中发现的成孔蛋白)，电子显微镜显示心肌细胞损伤可能与这些含有穿孔素的淋巴细胞有关。结果表明，在急性特发性和病毒性心肌炎中，心肌损伤可能是由于穿孔素分泌淋巴细胞的作用所致。

BACKGROUND & AIMS: :Rhizoxin is an antineoplastic drug that inhibits tubulin polymerization. In this study, we demonstrated that rhizoxin was approximately twice as active in vitro against a human small-cell lung cancer cell line with non-P-glycoprotein-mediated resistance to vindesine, H69/VDS, as against its parental line, H69. Tubulin polymerization in H69/VDS, demonstrated by Western blot analysis, was inhibited markedly by rhizoxin compared with that in H69, in a concentration-dependent manner. A drug-accumulation study showed that the intracellular rhizoxin level in H69/VDS was 15% lower than that in H69, whereas efflux from H69/VDS was enhanced slightly. These results indicate that enhanced inhibition of tubulin polymerization rather than increased intracellular drug concentration accounted for the higher sensitivity of H69/VDS to rhizoxin. In an experiment using mice with severe combined immunodeficiency and inoculated subcutaneously with H69/VDS, in vivo tumor growth was reduced markedly by three intermittent intraperitoneal doses of rhizoxin compared with that in mice inoculated with H69. Three weeks after the last rhizoxin dose, the relative treated/untreated tumor volumes were 0.29 for H69, but only 0.06 for H69/VDS, indicating that H69/VDS regrowth was minimal even after a 3-week treatment-free period. In conclusion, rhizoxin conquers vindesine resistance of a human small-cell lung cancer cell line in vitro and in vivo.

背景与目标： : 根瘤菌素是一种抗肿瘤药物，可抑制微管蛋白聚合。在这项研究中，我们证明了根瘤菌素在体外对具有非P-糖蛋白介导的对长春地辛H69/VDS的抗性的人小细胞肺癌细胞系的活性大约是其亲本H69的两倍。通过蛋白质印迹分析证明，与H69相比，根瘤菌素显着抑制了H69/VDS中的微管蛋白聚合，并具有浓度依赖性。药物积累研究表明，H69/VDS中的细胞内根瘤菌素水平15% 低于H69，而H69/VDS的流出略有增强。这些结果表明，对微管蛋白聚合的抑制作用增强而不是细胞内药物浓度的增加是H69/VDS对根瘤菌素的更高敏感性的原因。在使用具有严重联合免疫缺陷并皮下接种H69/VDS的小鼠进行的实验中，与接种H69的小鼠相比，通过三种间歇性的腹膜内剂量的根瘤菌素显着降低了体内肿瘤的生长。最后一次给药后三周，H69的相对治疗/未治疗的肿瘤体积0.29，但H69/VDS仅0.06，表明即使在3周的无治疗期后，H69/VDS的再生也很小。总之，根瘤菌素在体外和体内都能征服人类小细胞肺癌细胞系的长春地辛抗性。

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。