• 【神经元活动的同步促进单个大鼠新皮层神经元在早期发育中的存活。】 复制标题 收藏 收藏
    DOI:10.1111/j.1460-9568.1997.tb01449.x 复制DOI
    作者列表:Voigt T,Baier H,Dolabela de Lima A
    BACKGROUND & AIMS: Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro, beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co-activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.

    背景与目标: 神经活动被认为在大脑皮层发育过程中起着重要作用。在这项研究中,我们研究了整体活动阻滞或增强的影响以及图案化放电对培养的大鼠新皮层神经元在体外第二周 (突触开始后) 存活的能力的影响。通过添加河豚毒素 (TTX) 和增加培养基中的镁浓度来阻断神经元活性,从而大大降低了皮质细胞的存活。通过提高外部钾浓度来增加神经元活性,显着改善了皮质神经元的存活。我们推测,在发育中的神经元网络中,神经细胞的存活受到突触介导的事件的调节,这些事件涉及细胞内钙浓度的变化。为了进一步研究这个问题,我们通过同时光学记录许多神经元的细胞内钙信号来监测发育网络的活动。这些记录表明,在低镁的新皮层神经元中,其细胞内钙浓度表达同步振荡。在培养的第二周,网络使多个细胞的细胞内钙的变化同步的能力逐渐出现,同时突触密度增加和存活神经元数量减少。通过在记录过程几天后检查已识别细胞的命运,我们发现与其他神经元共同激活的神经细胞比不参与同步事件的细胞存活的机会要高得多。这些实验表明,在早期皮质网络发育过程中,皮质神经元显示出同步的放电活动,并且神经元的存活至少部分取决于这种神经元活动模式。
  • 【一系列赛庚胺类似物的合成,对5-HT2A,5-HT2B和5-HT2C 5-羟色胺受体的亲和力和结构-活性关系。】 复制标题 收藏 收藏
    DOI:10.1248/cpb.45.842 复制DOI
    作者列表:Honrubia MA,Rodriguez J,Dominguez R,Lozoya E,Manaut F,Seijas JA,Villaverde MC,Calleja JM,Cadavid MI,Maayani S,Sanz F,Loza MI
    BACKGROUND & AIMS: :Cyproheptadine is a drug that shows high affinity for type 2 (5-HT2) receptors. We studied a series of compounds obtained by modification of the tricyclic system of Cyp (dibenzocycloheptadiene): 2f (thioxanthene), 2g (xanthene), 2h (dihydrodibenzocycloheptadiene), 2j (diphenyl), 2i (fluorene), and 3b (phenylmethyl). Their activities at the rat cerebral cortex 5-HT2A receptor were (pKi +/- S.E.M.): 8.80 +/- 0.11 (Cyp), 8.60 +/- 0.07 (2f), 8.40 +/- 0.02 (2g), 8.05 +/- 0.03 (2h), 7.87 +/- 0.12 (2j), 6.70 +/- 0.02 (2i) and 6.45 +/- 0.02 (3b); those at the rat stomach fundus 5-HT2B receptor (pA2 +/- S.E.M.) were: 9.14 +/- 0.25 (Cyp), 8.49 +/- 0.07 (2f), 7.58 +/- 0.58 (2g), 7.02 +/- 0.14 (2h), 6.07 +/- 0.20 (2j), and undetectable (2i, 3b): and those at the pig choroidal plexus 5-HT2C receptor (pKi +/- S.E.M.) were: 8.71 +/- 0.08 (Cyp), 8.68 +/- 0.01 (2f), 8.58 +/- 0.20 (2g), 7.95 +/- 0.05 (2h), 7.57 +/- 0.04 (2j), 6.98 +/- 0.04 (2i) and 6.63 +/- 0.20 (3b). The slopes did not differ significantly from unity. The compounds exhibited the same order of activities at every type of receptor, and the most active molecules presented certain steric (butterfly conformation of the tricyclic system) and electrostatic (proton affinity on the top of the central rings) patterns. It is concluded that the activity of cyproheptadine derivatives at 5-HT2 receptors is related to these molecular features, which make feasible a common disposition to interact with all three 5-HT2 subtypes.
    背景与目标: : 赛庚胺是一种对2型 (5-HT2) 受体显示高亲和力的药物。我们研究了通过修饰Cyp (二苯并环庚二烯) 的三环体系获得的一系列化合物: 2f (噻吩),2g (xanthene),2h (二氢二苯并环庚二烯),2j (二苯基),2i (芴) 和3b (苯基甲基)。它们在大鼠大脑皮层5-HT2A受体上的活性为 (pKi +/-s.e.M.): 8.80 +/- 0.11 (Cyp),8.60 +/- 0.07 (2f),8.40 +/- 0.02 (2g),8.05 +/- 0.03 (2h),7.87 +/- 0.12 (2j),6.70 +/- 0.02 (2i) 和6.45 +/- 0.02 (3b); 大鼠胃底5-HT2B受体 (pA2 +/-s.e.M.) 为: 9.14 +/- 0.25 (Cyp),8.49 +/- 0.07 (2f),7.58 +/- 0.58 (2g),7.02 +/- 0.14 (2h),6.07 +/- 0.20 (2j) 和不可检测 (2i,3b): 猪脉络丛5-HT2C受体 (pKi +/-s.e.M.) 为: 8.71 +/- 0.08 (Cyp),8.68 +/- 0.01 (2f),8.58 +/- 0.20 (2g),7.95 +/- 0.05 (2h) 、7.57 +/- 0.04 (2j) 、6.98 +/- 0.04 (2i) 和6.63 +/- 0.20 (3b)。坡度与统一没有显着差异。这些化合物在每种类型的受体上都表现出相同的活性顺序,并且最具活性的分子呈现某些空间 (三环系统的蝴蝶构象) 和静电 (中心环顶部的质子亲和力) 模式。结论是,赛庚胺衍生物在5-HT2受体上的活性与这些分子特征有关,这使得与所有三种5-HT2亚型相互作用的共同处置是可行的。
  • 【注意缺陷多动障碍可能与中枢脑源性神经营养因子活性降低有关: 临床和治疗意义。】 复制标题 收藏 收藏
    DOI:10.1016/j.mehy.2006.06.025 复制DOI
    作者列表:Tsai SJ
    BACKGROUND & AIMS: :Attention-deficit hyperactivity disorder (ADHD) is a common childhood psychiatric disorder. Despite intensive research efforts, the aetiology of ADHD remains unknown. Current evidence suggests that the aetiology of ADHD is heterogeneous, comprising of multiple factors. Recently, it has been proposed that brain-derived neurotrophic factor (BDNF), a member of the neurotrophic factor family, may be implicated in the pathogenesis of ADHD. This hypothesis is supported by recent genetic studies in ADHD. Drawing on findings from studies into the drugs for ADHD relating to central BDNF expression, hyperactivity in BDNF knockout mice, BDNF effects in midbrain dopaminergic function and the close association between BDNF and the dopamine transporter (an important molecule for ADHD pathogenesis), it is proposed here that decreased central BDNF, particularly in the midbrain region, may play an important role in the pathogenesis ADHD. This hypothesis may have some implications for clinical findings in ADHD (for example, the co-morbidity between ADHD and major depression), and provide a new direction for the development of medication for ADHD treatment.
    背景与目标: : 注意力缺陷多动障碍 (ADHD) 是一种常见的儿童精神疾病。尽管进行了大量研究,但ADHD的病因仍然未知。目前的证据表明,ADHD的病因是异质的,由多种因素组成。最近,有人提出,神经营养因子家族的成员脑源性神经营养因子 (BDNF) 可能与ADHD的发病机理有关。该假设得到了ADHD最近的遗传研究的支持。根据对ADHD药物的研究结果,该药物与中枢BDNF表达,BDNF基因敲除小鼠的活动过度,BDNF在中脑多巴胺能功能中的作用以及BDNF与多巴胺转运蛋白 (ADHD发病机理的重要分子) 之间的密切联系有关,在这里提出降低中枢BDNF,特别是在中脑区域,可能在ADHD的发病机理中起重要作用。该假设可能对ADHD的临床发现 (例如,ADHD与重度抑郁症之间的合并症) 具有一定的意义,并为ADHD治疗药物的发展提供了新的方向。
  • 【具有NMDA拮抗剂活性的天然衍生肽抑制神经病理性疼痛。】 复制标题 收藏 收藏
    DOI:10.1016/s0006-8993(97)00183-2 复制DOI
    作者列表:Siegan JB,Hama AT,Sagen J
    BACKGROUND & AIMS: :Chronic pain may result from hyperexcitability following activation of spinal NMDA receptors. A naturally-derived mammalian peptide, histogranin, may possess NMDA antagonist activity. This study explored the possibility that stable analog [Ser1]Histogranin (SHG) could reduce chronic pain. Neuropathic pain was induced using the chronic constriction injury model (CCI). Intrathecal injection of SHG markedly attenuated the hyperalgesia and allodynia resulting from CCI, nearly normalizing responses. These results suggest that the natural peptide histogranin may be a novel adjunct in neuropathic pain management.
    背景与目标: : 脊髓NMDA受体激活后过度兴奋可能导致慢性疼痛。天然衍生的哺乳动物肽组织蛋白可能具有NMDA拮抗剂活性。这项研究探讨了稳定的类似物 [Ser1] 组织蛋白 (SHG) 可以减轻慢性疼痛的可能性。使用慢性收缩损伤模型 (CCI) 诱发神经病理性疼痛。鞘内注射SHG可显着减轻由CCI引起的痛觉过敏和异常性疼痛,几乎使反应正常化。这些结果表明,天然肽组织粒蛋白可能是神经性疼痛管理的新型辅助手段。
  • 【牙龈卟啉单胞菌主动调节 β2整合素粘附活性并促进与巨噬细胞的结合和内化。】 复制标题 收藏 收藏
    DOI:10.1128/IAI.00784-06 复制DOI
    作者列表:Hajishengallis G,Wang M,Harokopakis E,Triantafilou M,Triantafilou K
    BACKGROUND & AIMS: :In monocytes, the fimbriae of the oral pathogen Porphyromonas gingivalis activate cross talk signaling from Toll-like receptor 2 (TLR2) to the beta2 integrin CD11b/CD18, leading to the induction of the high-affinity state of the latter receptor. CD14 plays an important role in this "inside-out" proadhesive pathway by binding fimbriae and facilitating the activation of TLR2 and phosphatidylinositol 3-kinase signaling. In its high-affinity state, CD11b/CD18 mediates monocyte adhesion to endothelial cells and transmigration to sites of infection. We have now shown that P. gingivalis fimbriae function as both an activator and a ligand of CD11b/CD18; thus, fimbriae proactively promote their own binding to monocytes. Indeed, treatments that interfered with fimbria-induced activation of CD11b/CD18 (i.e., blockade of CD14, TLR2, or phosphatidylinositol 3-kinase signaling) also suppressed the cell binding activity of fimbriae, which was largely inducible and CD11b/CD18 dependent. Development of a recombinant inside-out signaling system in Chinese hamster ovary cells confirmed the ability of fimbriae to activate CD14/TLR2 signaling and induce their own CD11b/CD18-dependent binding. Induction of this proadhesive pathway by P. gingivalis fimbriae appeared to take place in lipid rafts. Indeed, methyl-beta-cyclodextrin, a cholesterol-sequestering agent that disrupts lipid raft organization, was found to inhibit the fimbria-induced assembly of CD14/TLR2 signaling complexes and the activation of the high-affinity state of CD11b/CD18. Experiments using macrophages from mice deficient in various pattern recognition receptors indicated that the receptors involved in the inside-out proadhesive pathway (CD14, TLR2, and CD11b/CD18) are important for mediating P. gingivalis internalization within macrophages. It therefore appears that P. gingivalis proactively modulates beta2 integrin adhesive activity for intracellular uptake.
    背景与目标: : 在单核细胞中,口腔病原体牙龈卟啉单胞菌的菌毛激活从Toll样受体2 (TLR2) 到 β2整合素CD11b/CD18的串扰信号,导致诱导后者受体的高亲和力状态。CD14通过结合菌毛并促进TLR2和磷脂酰肌醇3-激酶信号的激活,在这种 “由内而外” 的前粘附途径中起重要作用。在其高亲和力状态下,CD11b/CD18介导单核细胞与内皮细胞的粘附并转移到感染部位。我们现在已经证明,牙龈卟啉单胞菌菌毛既是CD11b/CD18的激活剂又是配体。因此,菌毛会主动促进其自身与单核细胞的结合。实际上,干扰菌毛诱导的CD11b/CD18激活 (即CD14,TLR2或磷脂酰肌醇3-激酶信号传导的阻断) 的治疗也抑制了菌毛的细胞结合活性,这在很大程度上是可诱导的并且CD11b/CD18依赖性。在中国仓鼠卵巢细胞中开发了重组的由内而外信号系统,证实了菌毛激活CD14/TLR2信号并诱导其自身CD11b/CD18-dependent结合的能力。牙龈卟啉单胞菌诱导这种前粘附途径似乎发生在脂质筏中。实际上,发现甲基-β-环糊精是一种破坏脂质筏组织的胆固醇螯合剂,可抑制菌毛诱导的CD14/TLR2信号复合物的组装以及CD11b/cd18高亲和力状态的激活。使用缺乏各种模式识别受体的小鼠的巨噬细胞进行的实验表明,参与由内而外的前粘附途径 (CD14,TLR2和CD11b/CD18) 的受体对于介导巨噬细胞内牙龈卟啉单胞菌内化很重要。因此,似乎牙龈卟啉单胞菌主动调节 β2整合素粘附活性以促进细胞内摄取。
  • 【用黄色荧光蛋白变体YFP-H148Q/I152L对碘化钠同向转运体活性的细胞成像。】 复制标题 收藏 收藏
    DOI:10.1152/ajpcell.00291.2006 复制DOI
    作者列表:Rhoden KJ,Cianchetta S,Stivani V,Portulano C,Galietta LJ,Romeo G
    BACKGROUND & AIMS: :The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS.
    背景与目标: : 碘化钠同向转运体 (NIS) 介导碘 (I(-)) 在甲状腺和其他组织中的转运,作为治疗靶标和核成像报告基因的重要性日益提高。目前使用放射性示踪剂和电生理技术测量体外的NIS活性。我们报告了使用I(-) 敏感且可遗传编码的黄色荧光蛋白 (YFP) 变体YFP-H148Q/I152L对NIS活性进行新型活细胞成像测定的开发。在稳定表达YFP-H148Q/I152L的FRTL-5甲状腺细胞中,I(-) 诱导细胞荧光的快速和可逆降低,其特征是1) 对细胞外I(-) (35 muM) 的高亲和力,2) NIS抑制剂高氯酸盐的抑制,3) 细胞外Na(+) 依赖性,和4) TSH依赖性,表明荧光变化是由于I(-) 通过NIS流入所致。FRTL-5细胞群内的单个细胞表现出NIS介导的I(-) 流入速率的3.5倍变化,说明YFP-H148Q/I152L用于检测NIS活性的细胞间差异。I(-) 还导致表达NIS的COS-7细胞中YFP-H148Q/I152L荧光的高氯酸盐敏感降低,但在缺乏NIS的细胞中不引起。这些结果表明,YFP-H148Q/I152L是基因转移后甲状腺细胞和非甲状腺细胞中NIS介导的I(-) 摄取的敏感生物传感器,并表明细胞I(-) 的荧光检测可能是研究的有用工具。NIS的病理生理学和药理学。
  • 【尿激酶型纤溶酶原激活剂和基质金属蛋白酶在大肠癌中的活性和表达。】 复制标题 收藏 收藏
    DOI:10.1186/1471-2407-6-211 复制DOI
    作者列表:Kim TD,Song KS,Li G,Choi H,Park HD,Lim K,Hwang BD,Yoon WH
    BACKGROUND & AIMS: BACKGROUND:Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues. METHODS:Cancer tissue samples were obtained from colon carcinoma (n = 12) and rectal carcinomas (n = 10). MMP-2 and MMP-9 levels were examined using gelatin zymography and Western blotting; their endogenous inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed by Western blotting. uPA, uPAR and PAI-1 were examined using enzyme-linked immunosorbent assay (ELISA). The activity of uPA was assessed by casein-plasminogen zymography. RESULTS:In both colon and rectal tumors, MMP-2, MMP-9 and TIMP-1 protein levels were higher than in corresponding paired normal mucosa, while TIMP-2 level in tumors was significantly lower than in normal mucosa. The enzyme activities or protein levels of MMP-2, MMP-9 and their endogenous inhibitors did not reach a statistically significant difference between colon and rectal cancer compared with their normal mucosa. In rectal tumors, there was an increased activity of uPA compared with the activity in colon tumors (P = 0.0266), however urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) showed no significant difference between colon and rectal cancer tissues. CONCLUSION:These findings suggest that uPA may be expressed differentially in colon and rectal cancers, however, the activities or protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, PAI-1 and uPAR are not affected by tumor location in the colon or the rectum.
    背景与目标:
  • 【快速荧光法测量pendrin (SLC26A4) Cl-/I-转运活性。】 复制标题 收藏 收藏
    DOI:10.1159/000095164 复制DOI
    作者列表:Dossena S,Rodighiero S,Vezzoli V,Bazzini C,Sironi C,Meyer G,Fürst J,Ritter M,Garavaglia ML,Fugazzola L,Persani L,Zorowka P,Storelli C,Beck-Peccoz P,Bottá G,Paulmichl M
    BACKGROUND & AIMS: :Malfunction of the SLC26A4 protein leads to Pendred syndrome, characterized by sensorineural hearing loss, often associated with mild thyroid dysfunction and goiter. It is generally assumed that SLC26A4 acts as a chloride/anion exchanger, which in the thyroid gland transports iodide, and in the inner ear contributes to the conditioning of the endolymphatic fluid. Here we describe a fast fluorometric method able to be used to functionally scrutinize SLC26A4 and its mutants described in Pendred syndrome. The validation of the method was done by functionally characterizing the chloride/iodide transport of SLC26A4, and a mutant, i.e. SLC26A4(S28R), which we previously described in a patient with sensorineural hearing loss, hypothyroidism and goiter. Using the fluorometric method we describe here we can continuously monitor and quantify the iodide or chloride amounts transported by the cells, and we found that the transport capability of the SLC26A4(S28R) mutant protein is markedly reduced if compared to wild-type SLC26A4.
    背景与目标: : SLC26A4蛋白的故障导致Pendred综合征,其特征是感音神经性听力损失,通常与轻度甲状腺功能障碍和甲状腺肿有关。通常认为SLC26A4充当氯化物/阴离子交换剂,在甲状腺中转运碘化物,在内耳中有助于调节内淋巴液。在这里,我们描述了一种快速荧光测量方法,该方法能够用于功能检查SLC26A4及其在Pendred综合征中描述的突变体。该方法的验证是通过功能表征SLC26A4的氯化物/碘化物转运和突变体即SLC26A4(S28R) 来完成的,我们先前在患有感音神经性听力损失,甲状腺功能减退和甲状腺肿的患者中进行了描述。使用我们在这里描述的荧光法,我们可以连续监测和定量细胞转运的碘化物或氯化物量,并且我们发现与野生型SLC26A4相比,SLC26A4(S28R) 突变蛋白的转运能力显着降低。
  • 【修饰的酶原作为蛋白水解酶的人工底物: 使用修饰的尿激酶原比色测定细菌胶原酶和基质金属蛋白酶的活性。】 复制标题 收藏 收藏
    DOI:10.1042/bj3230603 复制DOI
    作者列表:Verheijen JH,Nieuwenbroek NM,Beekman B,Hanemaaijer R,Verspaget HW,Ronday HK,Bakker AH
    BACKGROUND & AIMS: We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.

    背景与目标: 我们描述了一种评估所有类别蛋白水解酶活性的新原理,并展示了该原理在细菌胶原酶和人类基质金属蛋白酶 (MMPs) 定量测定中的应用。这一新原理的核心是存在一种原酶,该原酶可以通过一次蛋白水解事件被激活为活性酶。使用蛋白质工程技术将原酶中的常规激活序列替换为待确定的蛋白酶识别的人工序列。后者可以作为新改造的原酶的激活剂。在本文中,基于此原理描述了一种用于确定MMPs的简单比色测定法。借助蛋白质工程,已经制备了一种改良的尿激酶原,其中通常由纤溶酶识别的激活序列 (pro-Arg-Phe-Lys向上arrowIle-Ile-Gly) 已被预期被许多MMPs识别和水解的序列所取代 (Arg-Pro-Leu-Gly向上arrowIle-ile-Gly)。通过MMP激活修饰的尿激酶原而产生的活性尿激酶可以直接,使用尿激酶的特定显色肽底物进行测量,也可以通过尿激酶催化的纤溶酶原活化间接测量。测定对相等摩尔量的活性mmp的响应以MMP-2>MMP-9>MMP-1>MMP-3>MMP-7的顺序降低。MMP-9的检测限低于15μm,对应于3。每项测定75x10(-15) 摩尔。使用该测定法,类风湿关节炎患者的滑膜组织提取物与骨关节炎患者的滑膜组织提取物相比,以及与正常胃组织提取物相比,胃肿瘤提取物中的MMP活性增加。
  • 【N1-Benzoyl-N2-[1-(1-萘基) 乙基]-trans-1,2-二氨基环己烷: 4-氯苯基甲酰胺 (calhex 231) 作为新的钙感应受体配体的开发,证明了有效的钙分解活性。】 复制标题 收藏 收藏
    DOI:10.1021/jm051233+ 复制DOI
    作者列表:Kessler A,Faure H,Petrel C,Rognan D,Césario M,Ruat M,Dauban P,Dodd RH
    BACKGROUND & AIMS: :A structure-activity relationship (SAR) study was performed principally at the N1 position of N1-arylsulfonyl-N2-[1-(1-naphthyl)ethyl]-trans-1,2-diaminocyclohexanes, a new family of calcilytics acting at the calcium sensing receptor (CaSR). The most active compound in this series was the 4-(trifluoromethoxy)benzenesulfonyl derivative 7e, which displayed an IC50 of 5.4 +/- 0.5 microM with respect to the inhibition of calcium-induced tritiated inositol phosphate ([3H]IP) accumulation in Chinese hamster ovarian (CHO) cells expressing the CaSR. Replacement of the sulfonamide linkage of this compound by a carboxamide led to a 6-fold increase in activity (7m, IC50 = 0.9 +/- 0.2 microM). Among the carboxamides synthesized, one of the most active compounds was the 4-chlorophenylcarboxamide (1S,2S,1'R)-7n (Calhex 231, IC50 = 0.33 +/- 0.02 microM). The absolute configuration of (1S,2S,1'R)-7n was deduced from an X-ray crystallographic study of one of the diastereomers of compound 7d. The stereochemical preference for the (1S,2S,1'R)-isomers can be rationalized on the basis of a three-dimensional model of the calcilytic binding pocket of the CaSR. Removal of the C-1' methyl group or replacement of the 1-naphthyl group by a 2-naphthyl or biphenyl moiety led to appreciable loss of calcilytic activity. Compounds 7e, 7m, and Calhex 231 did not stimulate [3H]IP accumulation in CHO cells expressing or not expressing the CaSR.
    背景与目标: : 主要在N1-arylsulfonyl-N2-[1-(1-萘基) 乙基]-trans-1,2-二氨基环己烷的N1位置进行了构效关系 (SAR) 研究,作用于钙感应受体 (CaSR) 的新的钙离子家族。该系列中最具活性的化合物是4-(三氟甲氧基) 苯磺酰基衍生物7e,在表达CaSR的中国仓鼠卵巢 (CHO) 细胞中,对钙诱导的tri化肌醇磷酸 ([3H]IP) 积累的抑制作用显示出5.4/- 0.5微米的IC50。用甲酰胺代替该化合物的磺酰胺键导致6倍活动增加 (7m,IC50 = 0.9 +/- 0.2微米)。在合成的甲酰胺中,最具活性的化合物之一是4-氯苯基甲酰胺 (1S,2S,1'R)-7n (Calhex 231,IC50 = 0.33 +/- 0.02微米)。(1S,2S,1'R)-7n是根据对化合物7d的非对映异构体之一的x射线晶体学研究得出的。(1S,2S,1'R)-异构体可以在CaSR的煅烧结合袋的三维模型的基础上合理化。C-1甲基的去除或2-萘基或联苯部分取代1-萘基导致煅烧活性的明显损失。化合物7e,7m,并且Calhex 231不刺激表达或不表达CaSR的CHO细胞中的 [3H]IP积累。
  • 【冷暴露对大鼠肾上腺酪氨酸羟化酶的影响: RNA,蛋白质,酶活性和辅因子水平的分析。】 复制标题 收藏 收藏
    DOI:10.1111/j.1471-4159.1990.tb01232.x 复制DOI
    作者列表:Baruchin A,Weisberg EP,Miner LL,Ennis D,Nisenbaum LK,Naylor E,Stricker EM,Zigmond MJ,Kaplan BB
    BACKGROUND & AIMS: :Long-term cold exposure (5-7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3-6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48-72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold-induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre- and posttranslational regulation. Our findings also suggest that cold stress-induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.
    背景与目标: : 已知长期冷暴露 (5-7天) 会引起肾上腺髓质酪氨酸羟化酶 (TH) RNA,蛋白质和酶活性的同时增加。在本报告中,我们比较了这些变化的时间过程,并研究了冷暴露对酪氨酸羟基化所需的辅因子生物蝶呤水平的影响。冷暴露仅1小时后,与无应激对照相比,TH mRNA丰度71% 增加。在冷暴露的3-6小时内,总细胞TH RNA水平的增加最大 (是对照值的三倍),并且在整个实验期间 (72小时) 保持升高。冷暴露24小时后,TH蛋白水平迅速增加,并在48-72小时达到对照组的三倍的最大值。尽管TH RNA和蛋白质含量的升高相对较快且较大,但在冷暴露的最初48小时内仅检测到TH活性的适度增加。冷暴露开始后,肾上腺髓质生物蝶呤迅速增加,在24小时时升至非应激对照的大约两倍,并在整个应激期内保持在该水平。总之,该时间过程研究的结果表明,冷诱导的肾上腺TH活性的改变是由多种细胞控制机制介导的,其中可能包括翻译前和翻译后的调节。我们的发现还表明,冷应激引起的TH辅因子水平的增加可能是交感肾上腺系统对冷应激反应的另一个关键事件。
  • 【感知到的步行障碍。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Dunton GF,Schneider M
    BACKGROUND & AIMS: INTRODUCTION:Although the health benefits of walking for physical activity have received increasing research attention, barriers specific to walking are not well understood. In this study, questions to measure barriers to walking for physical activity were developed and tested among college students. The factor structure, test-retest and internal consistency reliability, and discriminant and criterion validity of the perceived barriers were evaluated. METHODS:A total of 305 undergraduate students participated. Participants had a mean age (+/- SD) of 20.6 (+/- 3.02) years, and 70.3% were female. Participants responded to a questionnaire assessing barriers specific to walking for physical activity. Perceived barriers to vigorous exercise, walking for transportation and recreation, and participation in lifestyle activities (such as taking the stairs instead of the elevator) were also assessed. Subsamples completed the walking barriers instrument a second time after 5 days in order to determine test-retest reliability (n = 104) and wore an accelerometer to measure moderate-intensity physical activity (n = 85). RESULTS:Factor analyses confirmed the existence of three factors underlying the perceived barriers to walking questions: appearance (four items), footwear (three items), and situation (three items). Appearance and situational barriers demonstrated acceptable reliability, discriminant validity, and relations with physical activity criteria. After we controlled for barriers to vigorous exercise, appearance and situational barriers to walking explained additional variation in objectively-measured moderate physical activity. CONCLUSION:The prediction of walking for physical activity, especially walking that is unstructured and spontaneous, may be improved by considering appearance and situational barriers. Assessing barriers specific to walking may have important implications for interventions targeting walking as means for engaging in physical activity.
    背景与目标:
  • 【甲基-β-环糊精的分析表征,具有减少Niemann-Pick病C1型细胞中溶酶体胆固醇积累的药理活性。】 复制标题 收藏 收藏
    DOI:10.1089/adt.2017.774 复制DOI
    作者列表:Li R,Hao J,Fujiwara H,Xu M,Yang S,Dai S,Long Y,Swaroop M,Li C,Vu M,Marugan JJ,Ory DS,Zheng W
    BACKGROUND & AIMS: :Methyl-β-cyclodextrin (MβCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease type C1 (NPC1) patient fibroblasts. However, the pharmacological activity of MβCD reported by different laboratories varies. To determine the potential causes of this variation, we analyzed the mass spectrum characteristics, pharmacological activity of three preparations of MβCDs, and the protein expression profiles of NPC1 patient fibroblasts after treatment with different sources of MβCDs. Our data revealed varied mass spectrum profiles and pharmacological activities on the reduction of lysosomal cholesterol accumulation in NPC1 fibroblasts for these three preparations of MβCDs obtained from different batches and different sources. Furthermore, a proteomic analysis showed the differences of these three MβCD preparations on amelioration of dysregulated protein expression levels in NPC1 cells. The results demonstrate the importance of prescreening of different cyclodextrin preparations before use as a therapeutic agent. A combination of mass spectrum analysis, measurement of pharmacological activity, and proteomic profiling provides an effective analytical procedure for characterization of cyclodextrins for therapeutic applications.
    背景与目标: : 甲基-β-环糊精 (m β cd) 减少Niemann-Pick病C1 (NPC1) 患者成纤维细胞中的溶酶体胆固醇积累。但是,不同实验室报道的m β cd的药理活性有所不同。为了确定这种变异的潜在原因,我们分析了三种m β cds制剂的质谱特征,药理活性以及用不同来源的m β cds处理后NPC1患者成纤维细胞的蛋白表达谱。我们的数据揭示了从不同批次和不同来源获得的这三种m β cds制剂在NPC1成纤维细胞中减少溶酶体胆固醇积累的不同质谱谱和药理活性。此外,蛋白质组学分析表明,这三种m β cd制剂在改善NPC1细胞中蛋白表达水平失调方面存在差异。结果表明,在用作治疗剂之前,对不同的环糊精制剂进行预检查的重要性。质谱分析,药理活性测量和蛋白质组学分析相结合,为表征用于治疗应用的环糊精提供了有效的分析程序。
  • 【再生大鼠肝脏线粒体H-ATPase活性和F1含量的变化。】 复制标题 收藏 收藏
    DOI:10.1016/0014-5793(85)80400-2 复制DOI
    作者列表:Buckle M,Guerrieri F,Papa S
    BACKGROUND & AIMS: :Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.
    背景与目标: : 在肝再生过程中从大鼠肝脏制备的亚线粒体颗粒表现出降低的ATPase活性,这与F1亚基含量的降低有关,如sdds-PAGE所示。使用针对H-ATPase复合物的F1部分的抗体表明,部分肝切除术后24小时,大鼠肝脏的线粒体颗粒和线粒体中F1的 β 亚基数量均明显减少。
  • 【NLRX1通过控制线粒体活性抑制组织损伤中的氧化应激和凋亡。】 复制标题 收藏 收藏
    DOI:10.1084/jem.20161031 复制DOI
    作者列表:Stokman G,Kors L,Bakker PJ,Rampanelli E,Claessen N,Teske GJD,Butter L,van Andel H,van den Bergh Weerman MA,Larsen PWB,Dessing MC,Zuurbier CJ,Girardin SE,Florquin S,Leemans JC
    BACKGROUND & AIMS: :Mitochondrial dysfunction is the most prominent source of oxidative stress in acute and chronic kidney disease. NLRX1 is a receptor of the innate immune system that is ubiquitously expressed and localized in mitochondria. We investigated whether NLRX1 may act at the interface of metabolism and innate immunity in a model of oxidative stress. Using a chimeric mouse model for renal ischemia-reperfusion injury, we found that NLRX1 protects against mortality, mitochondrial damage, and epithelial cell apoptosis in an oxidative stress-dependent fashion. We found that NLRX1 regulates oxidative phosphorylation and cell integrity, whereas loss of NLRX1 results in increased oxygen consumption, oxidative stress, and subsequently apoptosis in epithelial cells during ischemia-reperfusion injury. In line, we found that NLRX1 expression in human kidneys decreased during acute renal ischemic injury and acute cellular rejection. Although first implicated in immune regulation, we propose that NLRX1 function extends to the control of mitochondrial activity and prevention of oxidative stress and apoptosis in tissue injury.
    背景与目标: : 线粒体功能障碍是急性和慢性肾脏疾病中氧化应激的最突出来源。NLRX1是先天免疫系统的受体,广泛表达并定位在线粒体中。我们研究了NLRX1是否可能在氧化应激模型中作用于代谢和先天免疫的界面。使用嵌合小鼠肾缺血再灌注损伤模型,我们发现NLRX1以氧化应激依赖性方式保护死亡率,线粒体损伤和上皮细胞凋亡。我们发现NLRX1调节氧化磷酸化和细胞完整性,而NLRX1的丢失会导致缺血再灌注损伤期间的耗氧量增加,氧化应激以及上皮细胞的凋亡。我们发现,在急性肾缺血损伤和急性细胞排斥反应期间,人肾脏中NLRX1的表达降低。尽管首先涉及免疫调节,但我们建议NLRX1功能扩展到控制线粒体活性以及预防组织损伤中的氧化应激和凋亡。

+1
+2
100研值 100研值 ¥99课程
检索文献一次
下载文献一次

去下载>

成功解锁2个技能,为你点赞

《SCI写作十大必备语法》
解决你的SCI语法难题!

技能熟练度+1

视频课《玩转文献检索》
让你成为检索达人!

恭喜完成新手挑战

手机微信扫一扫,添加好友领取

免费领《Endnote文献管理工具+教程》

微信扫码, 免费领取

手机登录

获取验证码
登录