食管鳞癌组织中的端粒延长替代机制

目的 研究食管鳞癌组织中端粒酶逆转录酶(hTERT)蛋白和端粒延长替代(ALT)机制的表达,探讨其与食管癌关系.方法 应用免疫组织化学法,观察11例正常食管黏膜、12例癌旁伴有上皮不典型增生组织和64例食管鳞癌组织中早幼粒细胞白血病蛋白(PML)蛋白和hTERT蛋白的表达情况.结果 癌旁组织和癌组织PML蛋白阳性例数分别为1(8.33%)和10(15.62%),hTERT蛋白阳性率分别为41.67%(5/12) 和84.38%(54/64),两者hTERT蛋白阳性表达率存在显著差异(P＜0.01).结论 hTERT蛋白表达和ALT途径都可能存在于食管鳞癌组织中维持端粒长度.     

Polyions regulate the alternative amplification pathway of complement

Polyanion has previously been shown to inhibit generation of amplification pathway convertase of complement and to inhibit H-mediated decay of this complex. Polycations, protamine sulfate, and poly-l-lysine (PLL) were examined in this study for direct effects on alternative amplification pathway mechanisms and for ability to regulate polyanion-induced inhibiton; they were found to inhibit generation of EAC4b,3b,Bb, and EAC4b,3b,Bb,P in a dose-related manner. The generation of EAC4b,3b,B was also inhibited by higher doses but was augmented by lower doses of polycation. Polycation interfered with the effective consumption of B in the fluid phase, bound minimally to EAC4b,3b, and did not cause accelerated decay of a preformed convertase. Polycation diminished the ability of polyanion (heparin) to inhibit generation of both cell-bound and fluid-phase amplification pathway convertase. In contrast, polycation enhanced the ability of polyanion to cause decay of preformed convertase even though polycation had no effect itself. These studies demonstrate that both polyionic substances have the capacity to regulate amplification pathway mechanisms directly.     

Decay acceleration of the complement alternative pathway C3 convertase.

An ELISA-based method is described for analyzing the mechanism by which the decay of the alternative pathway C3 convertase is accelerated by C3 regulatory proteins. Using this assay, we show that human decay-accelerating factor (DAF) and factor H are active on mature convertase complexes (C3bBb) but not on their nascent precursor (C3bB). This finding has implications on the mechanisms of action of these two regulators. The complement convertases cleave the serum protein C3, and the resulting C3b activation fragments covalently attach to nearby targets where they direct antigen selection, immune clearance, and cell lysis. Several proteins, including the membrane protein DAF, and the serum protein factor H, limit convertase activity by promoting their irreversible dissociation. An understanding of the biochemical mechanisms providing for their activities would be helpful for the therapeutic control of the complement response.     

Caveolae--an alternative endocytotic pathway for targeted drug delivery

Caveolae are bottleshape-like invaginations of the plasma membrane. After internalization they are involved in endocytosis, transcytosis, potocytosis, and pinocytosis. Our recently expanded knowledge has made clear that various molecules and macromolecular complexes enter the cells after docking on caveolar receptors, before subsequent internalization of the caveolae. The internalization is initiated by the ligand receptor interaction. Folate, cholesterol, serum lipoproteins, and liposomes are among the most common examples. It is important to point out that, in contrast to the classic clathrin-dependent endocytosis, the caveolar internalization pathway seems to avoid the lysosomes. Internalized caveolae fuse with caveosomes, and the caveosomes deliver their contents into other subcellular (non-lysosomal) compartments. The bypass of the acidic and harmful milieu might be a major advantage for drug delivery via the caveolar pathway. Not all cell types have caveolae. Cells, where the Caveolin I protein is not expressed, do not develop caveolar invaginations. However, these cells have caveolar-equivalent plasma membrane domains, so-called "lipid rafts." Lipid rafts are assembled from the same lipid constituents and proteins as caveolae, but they are flat. Specific ligands may also dock on appropriate receptors of the raft domain. As a complication, certain types of ligand-covered raft receptors can migrate to clathrin-coated pits and get internalized via clathrin-coated vesicles. Nevertheless, suitable ligands or antibodies developed against proteins of the caveolar/raft domains may selectively direct the attached drug carrier to the less harmful caveolar/raft pathway. Thus, understanding of caveolar transport may help in the rational design of more effective drug carriers.     

补体旁路过度激活影响体内凝血功能

目的研究补体旁路的过度激活对体内纤溶、凝血及血小板的影响。方法采用尾静脉注射眼镜蛇毒因子(CVF)造成大鼠体内补体旁路的过度激活。检测大鼠给药前和给药后多个时间点血样的补体活性、t-PA、PAI-1、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(FIB)、P-selectin、PF-4、β-TG、血小板数量及聚集活性的变化,并检测小鼠静脉给予CVF后其尾部出血时间的变化。结果大鼠注射CVF后,血清补体活性在1h内基本消失。t-PA和PAI-1含量在1h内有明显增加,在48 h时间点t-PA/PAI-1比值有明显下降(P<0.01);PT、APTT、TT均略有变化,而血浆FIB含量明显下降;补体旁路的过度激活导致血中P-selectin、PF-4、β-TG明显上调,血小板数量明显减少,凝血酶和花生四烯酸诱导的血小板聚集被明显抑制,而ADP诱导的血小板聚集只受到轻微影响。小鼠尾部出血时间在1 h时明显缩短。结论补体旁路的过度激活能明显影响体内凝血功能,导致血液的高凝状态。     

Legal vulnerabilities in the treatment of chemically dependent dual diagnosis patients

Substance abusing patients with significant psychiatric disorders can present unique legal vulnerabilities for treatment professionals. The tendency within the chemical dependency community to treat patients uniformly can militate against the individualized treatment planning that satisfies legal obligations to act in the best interest of the patient. The failure to adequately consider both the psychiatric disorder and substance abuse problem may create legal vulnerabilities that issue from the failure to refer a patient in need of medication or the failure to prevent the suicide of a patient in a situation where suicide is preventable. Patients suffering harm from acts of commission or omission of a chemical dependency treatment specialist have recourse through laws regarding negligence. Variations in the backgrounds and training of care providers create problems for the harmed patient in establishing a standard of care for the treatment of chemical dependence. However, the psychological symptoms of the dual diagnosis patient make it easier to establish a standard of care that may be legally cognizable. The chemical dependency specialist--of whatever discipline--has a duty to patients that requires sound assessment and disposition.     

补体旁路激活导致内皮细胞活化和损伤

目的研究补体替代途径的激活对血管内皮细胞活化和损伤的作用。方法采用眼镜蛇毒因子(CVF)与人血清孵育,特异激活补体替代途径。将孵育物作用于人微血管内皮细胞,采用ELISA检测内皮细胞P-selectin、E-selectin、ICAM-1、MCP-1和IL-8的表达,采用酶活性测定法检测乳酸脱氢酶(LDH)活性,化学发光法检测内皮细胞caspase-8活化信号,MTT法检测内皮细胞增殖活性,并检测内皮细胞释放NO的变化。结果补体旁路激活导致内皮细胞瞬时表达P-selectin,并进而使内皮细胞上调表达E-selectin、ICAM-1、MCP-1和IL-8。内皮细胞经补体激活物刺激后,LDH释放增加、凋亡信号caspase-8活化上调以及NO释放下调,同时,细胞增殖也受到抑制。结论补体旁路激活能诱导内皮细胞活化和损伤,介导血管内皮的生理结构与功能发生变化,从而可能导致相应的炎症和组织损伤。     

小鼠血清补体替代途径溶血活性测定的新方法

目的针对现有补体溶血活性测定方法不适宜测定小鼠血清补体的缺点,构建一种血清用量少、灵敏度高的小鼠血清补体替代途径溶血活性测定的新方法。方法选用具有代表性的3个小鼠品系KM、BALB/c和C57BL/6小鼠的血清补体作为测定材料,以兔红细胞为溶血靶细胞,利用眼镜蛇毒因子(CVF)特异激活补体替代途径的特点,构建和优化小鼠血清补体替代途径溶血活性测定的方法,并采用该方法测定两种抗补体蛋白对小鼠体内血清补体活性的影响。结果以CVF作为激活剂成功构建了测定小鼠血清补体替代途径溶血活性的微量新方法。3个品系小鼠的血清在5～20μl的范围内即可分别达到合适的溶血度。该方法明显减少了血清用量,并能有效提高溶血度。结论基于CVF特异激活补体替代途径的特点而构建的小鼠血清补体替代途径溶血活性测定新方法,血清用量少、灵敏度高、稳定性好,适用于测定小鼠血清补体的溶血活性。     

氟喹诺酮类药物与其所致QT间期延长

近年来,氟喹诺酮类药物引致QT间期延长,从而诱发致死性尖端扭转型室性心动过速(torsade de pointes,Tdp)的心脏毒性日益引人注目.本文重点阐述了其发生机制、危险因素、不同药物的作用特点以及如何选用此类药物供临床参考.     

补体旁路激活致小鼠急性肺损伤的炎症病理机制研究

目的研究补体旁路激活所导致的小鼠肺部急性炎症的发生发展及相关指标的变化,为药物筛选及干预研究提供理想的小鼠肺部急性炎症病理模型。方法 SPF级昆明小鼠尾静脉注射眼镜蛇毒因子(CVF)激活血清补体旁路途径,根据注射后取样时间不同,分为15 min、30 min、1 h、2 h、6 h组,同时平行设置PBS对照组。取肺组织测定肺系数、肺含水量,并行病理切片检查,肺组织匀浆测定髓过氧化物酶(MPO)活性;制备支气管肺泡灌洗液(BALF)和血清,测定BALF中的细胞数和蛋白含量,采用ELISA法分别测定BALF和血清中的IL-6、TNF-α、P-selectin和ICAM-1含量。结果小鼠尾静脉注射CVF后可致肺部炎性细胞明显浸润,MPO活性明显上调,BALF中细胞总数和蛋白浓度明显增加。BALF和血清中的IL-6、TNF-α、P-selectin水平及血清中ICAM-1的含量均明显升高,其中,BALF中P-selectin含量在30 min时间点出现1个小高峰,IL-6和TNF-α在1 h时间点出现1个高峰,在2 h时间点均无进一步上升,但在6 h时间点各指标均又明显升高;血清中IL-6和TNF-α含量在1 h时间点达到峰值,随后浓度降低,P-selectin和ICAM-1水平随着时间的延长持续上升。而肺系数、肺含水量及BALF中ICAM-1的含量与PBS组相比无明显变化。结论补体旁路激活可导致小鼠肺部急性炎症的发生,以30 min至1 h的炎症反应最为明显,该实验可以为药物筛选及干预研究提供理想的动物肺部急性炎症病理模型。     

Highlighting the role of veterinary pharmacists in zoonotic diseases including COVID-19

Veterinary pharmacy is an often unknown and therefore, underrepresented career path for pharmacists. Uniquely, pharmacists—even untrained in veterinary medicine—are the only health professionals legally allowed to provide care for human and nonhuman patients. The 2019 coronavirus disease (COVID-19) pandemic is a peculiar situation that, not only highlights veterinary pharmacy as a career path, but stresses the role veterinary pharmacists, trained in both human and veterinary medicine, can play in zoonotic diseases. Specialized training in veterinary medicine allows the pharmacist to serve as a resource for both physicians as well as veterinarians during zoonotic events by helping to ascertain feasibility of therapeutic options given the species. In addition, veterinary pharmacists involved in translational research would be vital for the drug development process as they would be aware of biologic nuances between the species and how they may affect the ultimate therapeutic outcome.     

Biodegradation of dimethyl terephthalate by Pasteurella multocida Sa follows an alternative biochemical pathway

Pasteurella multocida Sa, a bacterial strain isolated from mangrove sediment by enrichment technique, was capable of transforming dimethyl terephthalate (DMT). Biodegradation of DMT was shown to take place as a series of sequential steps involving the hydrolysis of two ester linkages between the carboxyl groups of the terephthalate and the methyl side-chain initially to produce mono-methyl terephthalate (MMT) and then terephthalic acid (TA), respectively. However, with ethanol as the carrying solvent, there was a formation of one metabolite previously not observed. The two metabolites were characterized by high performance-liquid chromatography-electron ionization mass spectrometry as MMT and mono-ethyl terephthalate (MET), suggesting the existence of an alternative biochemical pathway in the degradation of DMT by P. multocida Sa. Since the presence of MMT and ethanol in culture inoculated with P. multocida Sa was prerequisites for the formation of MET, biologically mediated trans-esterification was proposed as a mechanism for the novel biochemical process observed.Special Issue on Biomarkers of Marine Pollution and Bioremediation     

Host recognition and target differentiation by factor H, a regulator of the alternative pathway of complement

Factor H is responsible for recognition of host cells and tissues and mediates discrimination among microbial pathogens during activation of the alternative pathway of complement (AP). Its unique structure of 20 SCR domains arranged in a flexible chain permits a variety of functional sites to interact with complement proteins and surface markers in a biological example of single-molecule combinatorial chemistry. In addition to the complement regulatory site located in the N-terminal four SCR domains, two other sites bind complement protein C3b and three sites appear to recognize a variety of polyanions that serve as host markers. Recent studies indicate that cooperativity among several C3b- and polyanion-binding sites influences the biological functions of factor H and that the degree of influence of each site varies on different cells. The engagement of one or more of the host marker recognition sites enables factor H to control activation of the AP. The absence of host-like markers allows AP activation, but many common pathogens have developed receptors for factor H or mimics of host markers of varying degrees of authenticity allowing them to escape detection by this innate defense system. Organisms using one or more of these evasive techniques include Neisseria gonorrhoeae, Streptococcus pyogenes, Yersinia enterocolitica, Trypanosoma cruzi, and the HIV virus.     

Characterization of potassium binding with human telomeres

Human telomeres are G‐rich tandem repeats that assume G‐quadruplex structures at the ends of chromosomes. Stabilization of telomeric G‐quadruplex represents a significant drug target for inhibiting the telomerase activity that is required in about 85% of cancers. Metal ions have been revealed as important stabilizers to DNA G‐quadruplexes, but their binding process with human telomeric G‐quadruplex remains unclear. In this report, we show that K⁺ traverses into the G‐tetrads centre of two G‐tetrad layers through the half‐capped top pathway constructed by the two edge‐wise loop bases. The binding is mediated by the electrostatic interactions between K⁺ and the nearby bases of G‐tetrads. However, direct traverse of K⁺ into the interior of G‐quadruplex is negatively regulated by the steric hindrance of water molecules. Once K⁺ enters the G‐quadruplex, stabilization of the in‐plane or sandwiched conformation of the telomeric G‐quadruplex?K⁺ complex is maintained by surrounding water molecules. These findings provide insights into the atomic interactions between K⁺ and telomere G‐tetrads for targeted drug design.     

Combining imaging and pathway profiling: an alternative approach to cancer drug discovery

Conventional drug discovery strategies are typically 'target centric' based on the selection of lead compounds with optimised 'on-target' potency and selectivity profiles. However, high-attrition rates are often the result of compensatory or redundant cancer mechanisms and the fact that tumours do not find it difficult to escape inhibition of a single pathway. In this article, we highlight two emerging and complimentary technologies; namely phenotypic imaging and post-translational pathway profiling, which when combined with relevant disease models can provide pharmacodiagnostic and drug combination strategies that predict and counteract inherent and adaptive drug resistance. The implementation of such approaches at early stages of the drug discovery process enables more informed decisions on candidate drug selection and how to maximise and predict efficacy before clinical development.