BACKGROUND & AIMS: We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.

背景与目标： 我们研究了苯巴比妥（PB）依赖和戒断大鼠大脑中谷氨酸受体的变化，立即早期基因的表达以及AP-1 DNA结合活性，以研究谷氨酸受体激活在PB戒断综合征中的可能。通过喂食药物混合的食物5周来制备PB依赖的大鼠。放射自显影分析表明，[3H（）-5-甲基-10,11-二氢-5H-二苯并[a，d]环庚烯-5,10-亚胺e（MK-801）与N-甲基- D-天冬氨酸（NMDA）受体，在PB依赖和24小时戒断大鼠的大脑皮层中显着增加。然而，在海马中的[3H] MK-801结合以及在海马和大脑皮层中的[3H] 6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX）和[3H]海藻酸结合基本上没有变化。组。 PB抽搐发作后，海马和大脑皮层中c-fos mRNA的表达增加，大脑皮层中c-jun mRNA的表达增加。通过施用MK-801抑制了c-fos和c-jun mRNA的诱导。此外，PB撤离可增强大脑中AP-1 DNA的结合活性。目前的发现表明，在PB戒断综合征的发展过程中，谷氨酸能神经传递的功能增强。

BACKGROUND & AIMS: The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.

背景与目标： GATA家族的脊椎动物DNA结合调节蛋白在不同的组织中以及在不同的发育时期表达。但是，这些蛋白质的DNA结合区具有相当的同源性，可以识别相当相似范围的DNA序列基序。 DNA结合是通过两个结构域介导的，每个结构域都包含一个锌指。先前的结果得出的结论是，尽管在某些情况下N末端指可以有助于结合的特异性和强度，但它不能独立地结合，而C末端指对于结合既是必需的又是足够的。在这里，我们表明，尽管对于GATA-1的N端手指来说确实如此，但GATA-2和GATA-3的手指却能够强烈独立地结合，并优先选择基序GATC。绑定需要在N末端指状体的两侧都存在两个基本区域。在GATA-1 N端手指附近缺少这些手指之一可能是其无法结合的原因。单个手指和两个基本区域的组合是一个基序的新变体，该变体先前已在其他手指蛋白的结合域中发现。我们的结果表明，N末端手指的DNA结合特性可能有助于区分GATA-2和GATA-3与GATA-1和其他GATA家族成员在体内的选择性调节作用。

BACKGROUND & AIMS: :Human class II MHC Ag are a family of cell surface glycoproteins. Their constitutive expression is limited to B lymphocytes and thymic epithelial cells. In many other cells their expression can be induced by IFN-gamma. Conserved upstream promoter sequences regulate this tissue-specific expression of class II genes. In the DRA promoter, one of these cis-acting regulatory motifs is the X2-box to which nuclear factor X2 (NF-X2) binds. Here, we present the isolation and characterization of the full-length cDNA clone encoding NF-X2. This cDNA clone was isolated by expression cDNA cloning, and encodes the human c-Jun protein, which together with c-Fos forms the heterodimeric activator protein-1 transcription complex. Whereas c-Fos/c-Jun heterodimers do not exist in B cells, they form and bind to the X2-box in class II nonexpressing cells. Thus, c-Fos/c-Jun heterodimers might contribute to the repression of DRA gene expression.

背景与目标： ：人类II类MHC Ag是细胞表面糖蛋白家族。它们的组成型表达仅限于B淋巴细胞和胸腺上皮细胞。在许多其他细胞中，它们的表达可以被IFN-γ诱导。保守的上游启动子序列调节II类基因的这种组织特异性表达。在DRA启动子中，这些顺式作用调控基元之一是X2-box，其与核因子X2（NF-X2）结合。在这里，我们介绍了编码NF-X2的全长cDNA克隆的分离和表征。该cDNA克隆通过表达cDNA克隆进行分离，并编码人c-Jun蛋白，该蛋白与c-Fos一起形成异二聚激活蛋白-1转录复合体。尽管B细胞中不存在c-Fos / c-Jun异二聚体，但它们在II类非表达细胞中形成并与X2-box结合。因此，c-Fos / c-Jun异二聚体可能有助于抑制DRA基因表达。

BACKGROUND & AIMS: :The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1alpha expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1alpha expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1alpha expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.

背景与目标： ：据报道肿瘤中的低氧环境在胰腺癌的进展中起重要作用。基质细胞与癌细胞之间的相互作用也有助于胰腺癌的恶性行为。在本研究中，我们调查了低氧刺激是否影响基质和胰腺癌细胞。我们的研究结果表明，缺氧显着提高了胰腺癌（PK8）和成纤维细胞（MRC5）中HIF-1alpha的表达。低氧刺激加速了PK8细胞的侵袭活性，因此，当用由低氧MRC5细胞制备的条件培养基（低氧条件培养基）培养低氧PK8细胞时，侵袭性进一步加快。在缺氧条件下，PK8细胞中的MMP-2，MMP-7，MT1-MMP和c-Met表达增加。缺氧刺激还增加了MRC5细胞的肝细胞生长因子（HGF）分泌，这导致PK8细胞中c-Met磷酸化的升高。相反，通过从低氧条件培养基中除去HGF，可以降低PK8细胞的癌浸润，MMP活性和c-Met磷酸化升高。在免疫组织化学研究中，在周围的基质细胞和胰腺癌细胞中均观察到了HIF-1alpha的表达，因此表明在癌细胞和基质细胞中均存在缺氧。此外，发现基质HGF表达不仅与癌细胞中的基质HIF-1α表达而且与c-Met表达显着相关。这些结果表明基质以及癌细胞内的低氧环境激活了HGF / c-Met系统，从而促进了胰腺癌的侵袭性侵袭性特征。

BACKGROUND & AIMS: :Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PuID required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.

背景与目标： ：相关的外膜蛋白，称为促胰液素，参与许多革兰氏阴性细菌的外膜大分子的分泌。在支链淀粉酶分泌系统中，PulS是一种与外膜相关的脂蛋白，是PulD促胰液素的完整性和正确的外膜定位所必需的。在这里，我们显示PulS结合位点位于PulD的C末端65个残基内。此结构域添加到丝状噬菌体分泌蛋白，pIV或无关的麦芽糖结合蛋白上，使得这两种蛋白都依赖于PulS来保持稳定性。由噬菌体f1 pIV和PuID的C末端结构域组成的嵌合蛋白需要正确定位PulS以支持噬菌体装配。通过共免疫沉淀法和亲和色谱法检测到了pIV-PulD65嵌合体和PulS之间形成的体内复合物。

BACKGROUND & AIMS: MyristoylCoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins.

Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

背景与目标： MyristoylCoA ：蛋白N-肉豆蔻酰基转移酶（NMT）催化稀有细胞脂肪酸肉豆蔻酸酯与多种真核蛋白N-末端Gly残基的共翻译共价连接。肉豆蔻酰基部分通常对于表达这些蛋白质的生物学功能是必不可少的。

仅C14 ：0的附件仅提供了不足以使与膜稳定结合的疏水性。因此，N-肉豆蔻酰蛋白的分区通常由通过附加的共价或非共价修饰起作用的“开关”调节。白色念珠菌是免疫力低下的人体内全身真菌感染的主要原因，它包含一个对其生存能力至关重要的单个NMT基因。人和白色念珠菌NMT的酰基辅酶A结合位点的功能特性非常相似。但是，它们的肽结合位点存在明显差异。 ADP核糖基化因子（Arf）包括在真菌酶的几种细胞蛋白底物中。来自N末端Arf序列（GLYASKLS-NH2）的八肽的丙氨酸扫描诱变显示，Gly1，Ser5和Lys6在结合中起主要作用。 ALYASKLS-NH2是一种对肽[Ki（app）= 15.3 /-6.4 microM]具有竞争性的抑制剂，对肉豆蔻酰辅酶A不具有竞争性。值得注意的是，用11-氨基十一烷酰基取代N末端四肽会产生竞争性抑制剂（11-氨基十一烷酰基-SKLS-NH2），其效力比[Ki（app）= 0.40 /-0.03 microM]高40倍左右。起始八肽。从C末端去除Leu-Ser会产生竞争性的二肽抑制剂（11-氨基十一烷酰基-SK-NH2），Ki（app）为11.7 /-0.4 microM，与起始八肽相当。含有C-末端N-环己基乙基赖氨酰胺部分的衍生物二肽抑制剂具有更有效的优势（IC 50 ＝ 0.11 /0.03μM），并且对细胞羧肽酶的消化具有抵抗力。刚性化柔性氨基十一烷酰基链会产生非常有效的常规NMT抑制剂（IC50 = 40-50 nM）。用2-甲基咪唑代替N端胺，并在刚性元素中添加具有R立体化学的苄基α-甲基，可产生更强效的抑制剂（IC50 = 20-50 nM），对真菌的选择性高达500倍与人类酶相比。该系列化合物的一个相关的效力较弱的成员是抑真菌的。使用细胞Arf作为报告基因判断，其生长抑制作用与细胞蛋白N-肉豆蔻酰化的减少有关。这些研究确定NMT是一种新的抗真菌靶标。