结核分枝杆菌katG Asn329Val突变导致异烟肼耐药

摘要：目的 研究结核分枝杆菌katG基因Asn329Val突变导致异烟肼耐药的机制。方法 扩增含有突变位点和野生型的全长katG基因,经TA克隆、定向克隆构建表达载体,原核表达KatG蛋白,纯化后比较重组KatG酶活性。结果 获得了katG基因的高效表达。重组蛋白酶活性显示,Asn329Val突变导致重组KatG过氧化氢酶活性完全丧失,但仍保持一定的过氧化物酶活性;Ser315Thr突变导致重组KatG过氧化氢酶和过氧化物酶活性部分丧失;Arg463Leu突变对过氧化氢酶活性和过氧化物酶活性影响均不大。结论 katG Asn329Val突变引起了KatG过氧化氢酶活性的完全丧失,可能是导致菌株对异烟肼耐药的机制。     

结核分枝杆菌katG基因突变对异烟肼耐药性的影响

目的探讨结核分枝杆菌katG基因突变TGG328TTT(Trp328Phe)对异烟肼耐药性的影响。方法以结核分枝杆菌标准株H37Rv和临床突变株DNA为模板,采用PCR扩增野生型和双突变型(含Trp328Phe与Met420Thr)katG基因,采用重叠延伸PCR技术构建单突变型(Trp328Phe)katG基因。将各katG基因重组至质粒pET22b(+),再导入表达宿主菌BL21(DE3)pLySs中,IPTG诱导目的蛋白表达,亲和层析法对重组KatG蛋白进行分离纯化。通过各重组KatG蛋白与过氧化氢和邻联大茴香胺反应,检测受试菌过氧化氢酶和过氧化物酶的活性。结果成功获得纯化的KatG蛋白。相比野生型KatG(19.05U/mg和7.05×10-3 U/mg),单突变型KatG(10.50U/mg和2.23×10-3 U/mg)过氧化氢酶和过氧化物酶活性分别下降44.9%和68.3%,双突变型KatG(11.88U/mg和3.31×10-3 U/mg)则分别下降37.6%和53.0%。结论 katG基因(Trp328Phe)突变引起KatG酶活性部分缺失,与异烟肼耐药有关,可作为耐药菌株基因检测的标志用于基因芯片的开发。双突变时KatG蛋白活性较高,提示katG-328TTT具有代偿性突变的作用。     

耐多药结核分枝杆菌异烟肼耐药相关基因katG突变分析

异烟肼(isoniazid, INH)是一种重要的抗结核药物,其在细菌体内被过氧化氢酶-过氧化物酶KatG氧化为乙酰异烟肼自由基和活性氧自由基等活性物质,进而攻击结核分枝杆菌多个靶位点来发挥杀菌作用。Zhang等通过Southern印迹杂交（Southern blot）发现结核分枝杆菌katG基因缺失和异烟肼耐药相关。研究表明,异烟肼耐药与许多基因相关,发生频率最高的为katG编码区和inhA启动子区的突变,katG315位突变最常见,有30%～94%的耐药株发生该突变,但国内对于该基因其他位点的突变研究较少。本研究通过对河南省耐多药(multidrug resistant,MDR)结核分枝杆菌katG编码区全长突变进行分析,进一步分析异烟肼的耐药机制。     

高分辨率熔解曲线技术用于结核分枝杆菌临床分离株异烟肼耐药性的快速检测

目的评价高分辨率熔解曲线分析技术(high-resolution melting curve,HRM)检测结核分枝杆菌异烟肼耐药性的应用价值。方法 1)通过传统比例法药敏实验对本实验室保存的49株结核分枝杆菌进行异烟肼耐药性分析。2)进一步对该结核分枝杆菌进行异烟肼耐药相关基因KatG基因和inhA基因进行异烟肼耐药决定区测序分析,筛查突变位点。3)根据筛查到的突变位点设计高分辨率熔解曲线分析所用的特异性引物,对异烟肼耐药基因耐药决定区进行高分辨率熔解曲线分析检测DNA突变,评估用高分辨率熔解曲线分析技术对结核分枝杆菌异烟肼耐药性检测的效率。结果 1)比例法药敏结果显示,49株实验菌株中20株为异烟肼耐药株,29株为异烟肼敏感株。2)测序分析结果显示:(1)KatG基因出现了4种突变形式,分别是234单位点突变;234、315双位点突变;234、463双位点突变;234、315、463三位点联合突变。(2)inhA基因检测到3种突变,即-8位、-15位、-152位。3)(1)对耐药株的基因突变进行分析发现:20株异烟肼耐药株中11株存在KatG基因第315位密码子的突变、占异烟肼耐药株的55%;6株存在inhA基因-15(4株)位碱基、-8(1株)位碱基、-152(1株)位碱基的突变,共占异烟肼耐药株的30%;2株同时存在基因KatG315密码子和inhA-15位碱基的突变,占异烟肼耐药株的10%;1株均未检测到KatG基因和inhA基因的突变,占异烟肼耐药株的5%。通过基因突变检测结核分枝杆菌异烟肼耐药性的敏感性为95%、特异性为100%。(2)用高分辨率溶解曲线检测实验菌株耐药基因突变的结果显示,18株存在基因突变,24株不存在基因突变,检测的灵敏度为94.7%、特异性为80%。(3)以高分辨率熔解曲线检测到突变为耐药的判断标准,检出19株为耐药株,24株为敏感株。以比例法药敏结果为参照,检测的灵敏度为95%、特异性为82.76%。结论高分辨率溶解曲线用于结核分枝杆菌对异烟肼耐药性的检测具有较好的灵敏度,耗时短,在异烟肼耐药结核病的快速诊断方面具有一定的应用价值。     

利用荧光偏振技术检测耐异烟肼结核分枝杆菌KatG 315点突变

目的 应用模板指导的荧光染料终止物掺入反应-荧光偏振检测技术(templatedirected dye-terminator incorporation with fluorescence polarization detection，TDI-FP)分析结核分枝杆菌KatG 315点突变与异烟肼耐药性的关系，并建立一种准确、快速的诊断结核分枝杆菌异烟肼耐药性的新方法。方法 对临床82例耐多药结核病患者所感染的结核分枝杆菌菌株进行常规培养和药敏实验；提取结核分枝杆菌DNA，并经聚合酶链反应(PCR)扩增271bp的KatG基因片段，PCR产物经消化处理清除残余dNTPs和引物，进行TDI反应，应用Victor2测定荧光偏振值，分析所有样品的KatG基因315位点的基因型。结果 29例异烟肼高度耐药患者的结核分枝杆菌中有15例发生KatG 315的G→C突变，其中4例为KatG 315突变和未突变的混合感染，突变率为52％；32例异烟肼低度耐药患者的结核分枝杆菌中有15例为KatG 315突变和未突变的混合感染，突变率为47％；21例异烟肼敏感患者的结核分枝杆菌未发现KatG 315突变。结论 KatG 315突变与结核分枝杆菌对异烟肼产生耐药性密切相关；应用TDI-FP技术检测KatG 315突变具有准确、快速、简便以及高通量等优点，在结核分枝杆菌耐异烟肼的临床诊断中有着广阔的应用前景。     

结核分枝杆菌katG S315T突变频率及突变株特征的研究

近年来分子遗传学研究表明，异烟肼（INH）耐药机制复杂，涉及多个基因，至少包括katG、inhA、kasA以及ahpC等基因。研究表明，INH耐药与编码过氧化氢酶．过氧化物酶的katG基因突变的联系最强，其中katG基因的一种特定替换，315位密码子的AGC→ACC（Ser→Thr，或katG S315T）颠换报道最多。该突变可降低INH耐药菌株中过氧化氢酶活性，但可保持足够水平的该酶的过氧化物酶活性，后者为细菌解毒所必需。世界各地结核分枝杆菌菌株中，katG S315T突变的流行率一般为26．0％～93．6％，结核病高发区流行率高。在俄罗斯圣彼德堡地区，该突变还与耐多药性有关，在荷兰该突变亦与高水平耐药、耐其他药物有关。研究我国农村耐多药结核病（MDR-TB）高发地区INH耐药分离株中katG S315T突变的发生频率与突变株特征有利于耐药结核病的防治。     

结核分枝杆菌异烟肼耐药基因katG的检测

目的探讨结核分枝杆菌异烟肼(INH)耐药的分子机制,建立快速检测结核分枝杆菌INH耐药性的方法。方法应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测105株结核分枝杆菌临床分离株的katG基因,运用DNA测序和生物信息分析比对进行验证。结果以H37Rv标准株为对照,34株敏感株中5株(14.7%)存在KatG基因异常,105株耐药株中55株(52.4%)存在KatG基因异常。结论结核分枝杆菌对INH耐药与KatG基因突变有关,PCR-RFLP技术可快速、准确地检测结核分枝杆菌对INH的耐药性。     

结核分枝杆菌KatG-ACT271CCT(Thr271Pro)突变与异烟肼耐药关系的研究

目的 探讨结核分枝杆菌katG ACT271CCT(Thr271Pro)基因点突变对异烟肼耐药性的影响方法 应用基因克隆技术合成野生型katG、突变型katG(AGC315ACC)和katG(ACT271CCT)基因片段,分别重组至质粒pET22b(+),转入表达菌BL21(DE3)pLySs,IPTG诱导目的蛋白表达,亲和层析法对目的蛋白分离纯化。最终检测重组katG蛋白过氧化物酶及过氧化氢酶活性。此外,通过软件AutoDock4.0采用半柔性对接方式分别模拟野生型katG蛋白、Thr271Pro突变型katG蛋白与异烟肼相互作用结果 成功获得纯化katG重组蛋白。相比野生型katG蛋白(过氧化氢酶活性361.5 U/mg和过氧化物酶活性77.6 U/mg),Thr271Pro突变型katG蛋白过氧化氢酶和过氧化物酶活性分别下降56%和63%(158.8 U/mg和28.7 U/mg),而Ser315Thr突变型katG蛋白过氧化氢酶和过氧化物酶活性分别下降74%和75%(96.2 U/mg和19.7 U/mg)。分子对接结果提示,异烟肼与Thr271Pro突变型katG蛋白结合自由能较野生型升高,Thr271Pro突变蛋白与异烟肼结合的亲和性降低。271位氨基酸突变后, Thr271Pro突变型katG蛋白氧化反应活性空腔发生一定的变形结论 katG Thr271Pro基因突变引起katG蛋白酶活性部分缺失和氧化反应活性空腔发生变形,导致异烟肼耐药。     

定向诱变方法研究结核分支杆菌KatG基因突变与异烟肼耐药机制的关系

目的：探讨结核分支杆菌KatG基因的常见点突变是否会导致与产生异烟肼耐药性相关的过氧化氢酶活性降低。方法：用定向诱变方法产生耐异肼的结核分支杆菌中最常见的KatG基因315突变体,造成该位点从丝氨酸（AGC）到苏氨酸（ACC）的突变,随后构建含KatG基因S315T突变体的质粒,转化进入大肠菌并实现高表达,对表达的蛋白进行过氧化氢酶活性的测定。结果：通过定向诱变方法成功获得KatG基因S315T突变体,并通过pET24b质粒转入大肠杆菌,KatG基因S315T突变体蛋白在大肠杆菌中得到高表达,对DatG S315T表达产物的过氧化氢酶活性进行检测,发现比野生株KatG基因表达产物酶活性下降了50％左右。结论：KatG基因315位从丝氨酸（AGC）到苏氨酸（ACC）的突变造成过与异烟肼耐药产生直接相关的氧化氢酶活性的下降。     

堪萨斯分枝杆菌临床分离株对异烟肼敏感性差异机制研究

目的:测定堪萨斯分枝杆菌临床分离株对异烟肼的敏感性，并探讨引发其差异的原因。方法:结核分枝杆菌标准株H37Rv(ATCC27294)、堪萨斯分枝杆菌标准株(ATCC12478),以及35株分离自非结核分枝杆菌感染患者的堪萨斯分枝杆菌临床分离株均来源于首都医科大学附属北京胸科医院。微孔板法测定35株堪萨斯分枝杆菌临床分离株加入外排泵抑制剂(氰化羰基-3-氯苯腙、二环己基碳二亚胺、维拉帕米和利血平)前后的异烟肼体外最低抑菌浓度(minimum inhibitory concentration, MIC)变化，并评估其关联性；测定已知与异烟肼耐药相关的基因序列，了解基因序列多态性与MIC值的相关性。过表达结核分枝杆菌和堪萨斯分枝杆菌过氧化氢-过氧化物酶(KatG),利用生物层干涉技术检测蛋白与异烟肼亲和力，利用酶联免疫法检测蛋白过氧化物-过氧化氢酶活性。结果:35株堪萨斯分枝杆菌临床分离株中，异烟肼对5.7%(2/35)、71.4%(25/35)、2.9%(1/35)和20.0%(7/35)的菌株的MIC值分别为0.5、1、4和>32μg/ml;分别加入适量氰化羰基-3-氯苯腙、二环己...     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.