我国新发现的鼠疫自然疫源地鼠疫菌基因组序列测定及分析

目的 测定和分析我国新发现的鼠疫自然疫源地鼠疫菌株（耶尔森菌）的全基因组序列,探讨玉龙疫情菌株(D106004)与邻近两个疫源地剑川菌株(D182038)和西藏菌株(Z176003)的亲缘关系。方法 采用全基因鸟枪法及Solexa方法对3株鼠疫菌株进行全基因组测序,并进行比较基因组学分析。基因组间编码序列比较分析采用BLAST软件进行,基因组间重排分析采用MAUVE软件进行。结果 鼠疫菌株D106004、D 182038、Z176003均具有1个染色体和3个质粒,菌株间染色体、质粒特征基本相似;3株菌株间编码序列的蛋白相邻类的聚簇(COG)功能分类及插入序列数目比较差异无统计学意义(x2值分别为3.03、0.257,P均＞0.05)。菌株间编码序列、单核苷酸多态性(SNPs)和基因组重排结果显示,在3株菌株中,有2882个基因具有100％的同源性。其中D106004菌株预测的3636个基因中与D182038菌株一致的基因有2994个,90％以上相似的基因有240个;与Z176003菌株一致的基因有3113个,90％以上相似的基因有200个;D106004菌株与Z176003、D182038菌株的同义SNPs数为59、68个,非同义SNPs数为104、203个;D106004与Z176003菌株之间可分为11个重排片段,较之D106004与D182038菌株之间的16个重排片段数目明显减少。结论 3株鼠疫菌株基因之间具有高度同源性,D106004与Z176003菌株之间的亲缘关系较之与D182038菌株更为接近,玉龙疫源地菌株可能是由西藏疫源地菌株进化而来。     

鼠疫耶尔森菌基因组中IS100插入位点的特异性分析

目的 通过鼠疫耶尔森菌基因组中插入序列IS100位点的特异性研究,对我国鼠疫菌基因组的构成类型和特点进行分析.方法 以鼠疫菌东方变种CO92全长序列为参考,选择5个位置的IS100位点,在IS100基因相邻两外侧设计1对引物,在IS100基冈内设汁1对向外引物使其分别能够与两外侧引物扩增.对来自13个生态型的91株鼠疫菌和1株假结核耶尔森菌进行PCR扩增,产物进行克隆测序分析,同时把各菌株的IS100位点情况标记在我国鼠疫疫源地类型慨图上二进行分析.结果 实验所用91株鼠疫菌在所选定IS100位点出现含有和不含IS100的插入序列,基因片段组成相对CO92序列已发生改变,而且这些IS100基因型的组成在我国鼠疫自然疫源地中呈现区域性分布.结论 IS100基因型别分布与我国鼠疫菌疫源地分型分布基本一致,并直接反映了鼠疫菌基因组某特定位点的基因组成状况及其在进化过程中的高度流动性.     

应用标识基因检测鼠疫菌的研究

英国Sanger研究中心、美国Wisconsin大学和我国分别于2001,2002和2004年完成了鼠疫耶尔森氏菌CO92,KIM,91001的全基因组序列测定工作[1～3],这有助于研究鼠疫菌的进化过程,为寻找新的检测手段提供了便利研究条件.聚合酶链反应(PCR)自1985年发明至今,经过不断开发和改进,现已经广泛应用在诸多领域,是分子生物学、临床诊断、法医检验实验室的常规技术.我们采用我国91001菌株序列上YPO2088标识基因(编码甲基化转移酶)[4],对150份样本进行PCR扩增,现将结果报道如下.     

利用分子生物学技术对鼠疫耶尔森菌的分类鉴定

目的 对4株可疑菌株进行系列验证实验,以确定其是否为鼠疫耶尔森菌(以下简称鼠疫菌)自然分离株.方法 用鼠疫细菌学常规方法和分子生物学手段确定其生物学表型特征、特异性基因及基因组特征.结果 4株菌具备鼠疫菌的典型形态特征;能被鼠疫噬菌体完全裂解;主要生化特性为阿胶糖(+)、鼠李糖(-)、麦芽糖(+)、蜜二糖(-)、甘油(-)、脱氮(+),与鼠疫菌同源菌株菌苗株(EV76)一致;其毒力因子为F1(+),VW(+),Pgm(-),PstⅠ(+),与鼠疫菌菌苗株(EV)特征完全一致;109个/ml可疑菌对实验动物小白鼠无致死能力.具有鼠疫标识基因;差异片段(DFR)分型结果,4株菌的24个DFR扩增,DFR谱不同于任何鼠疫菌自然分离菌株基因组型.结论 尽管4菌株具备鼠疫菌自然分离株的一些表型特征,但基因组特征表明其不是鼠疫菌自然分离株,而是鼠疫菌同源菌株菌苗株(EV菌).     

随机扩增多态性DNA技术检测不同地区田鼠型鼠疫耶尔森氏菌基因结构

目的　了解不同地区田鼠型鼠疫耶尔森氏菌 (以下简称鼠疫菌 )之间的亲缘关系和基因类型。方法　随机扩增多态性DNA(RAPD)技术。结果　扩增的 13株田鼠型鼠疫耶尔森氏菌在凝胶电泳所显示的条带 ,除 3株菌缺少部分条带外 ,其余菌株基本相同。结论　青海田鼠型鼠疫耶尔森氏菌和布氏田鼠型鼠疫耶尔森氏菌具有相似性 ,在遗传学上属于同源。     

陕西省两起鼠间鼠疫流行鼠疫菌生物学特性比较

目的通过对两起鼠间鼠疫流行鼠疫菌生物学特性进行比较,了解陕西省动物间鼠疫流行的病原特点。方法对2000～2001年、2006年动物鼠疫流行期间所分离菌株的生化、毒力、毒力因子及质粒进行比较分析。结果被鉴定菌株发酵阿胶糖,分解甘油,不发酵鼠李糖、麦芽糖,脱氮阴性。所有测试菌株含有F1和Pst1因子,2000～2001年19株鼠疫菌除1株Pgm±外,均含有4种毒力因子,2006年5株测试鼠疫菌均不含VW因子,2株不含Pgm因子。2000～2001年5株测试菌LD50在41～180个菌之间,2006年5株测试菌LD50在100～12.5亿个菌之间;2000～2001年未进行质粒检出工作,2006年鼠疫菌检出6、45、65 MD 3种质粒。结论所有鉴定菌株生化特点符合鄂尔多斯高原沙鼠鼠疫菌生化特性,引起两起鼠间鼠疫的鼠疫菌毒力因子及毒力不完全相同,2000～2001年鼠疫菌为强毒鼠疫菌,2006年鼠疫菌毒力有强弱之分,5株测试菌3株强毒菌,2株弱毒菌。     

云南省家鼠疫源地鼠疫耶尔森氏菌分子流行病学特征研究北大核心CSCD

目的探讨云南家鼠疫源地鼠疫菌在云南鼠疫菌遗传进化上所处的位置,及其种群流行演化规律。方法按照不同地点、时间及分离源随机选取186株家鼠疫源地鼠疫菌,采用差异区段(DFR)、规律成簇的间隔短回文重复序列(CRISPRs)及多位点可变数目串联重复序列分析(MLVA)3种方法进行分子分型分析;以DFR+CRISPRs双重方法聚类分析划分基因簇,以MLVA26对基因簇再次聚类分析划分亚簇。结果 186株家鼠疫源地鼠疫菌中的184株聚为一个簇(家鼠鼠疫簇),其余2株为独立株;近史流行期、复燃流行期和2016年疫情分离株分别处于不同的亚簇,它们之间的位点差异至少有5个;复燃流行期菌株共有5个亚簇和6个独立株,其中2个亚簇为主要的基因亚簇,即滇西亚簇和滇西南-滇东-广西-贵州亚簇,它们之间有4个位点差异。结论家鼠鼠疫菌是云南鼠疫菌中最晚出现的菌株,近史流行期菌株与复燃流行期菌株菌株存在较大差异,而复燃流行期的鼠疫流行是复燃和扩散并存的结果。     

青海省乌兰县鼠疫病原学分析及流行病学意义

目的分析青海省乌兰县鼠疫菌株生物学特性及流行病学意义,为该地区的鼠疫防治提供科学依据。方法对1966—2011年青海省乌兰县分离的65株鼠疫菌株进行生化试验、毒力测定、毒力因子鉴定、质粒分析、鼠疫菌差异区段(Different Region,DFR)分型等研究。结果 65株被试菌株生物型为古典型、生态型均为青藏高原型。菌株基因组型为8型、5型、1b型、37型、44型,主要基因组型为8型。携带3种质粒谱,83.08%(54/65)菌株携带分子质量为6×106、45×106、52×106的质粒谱。93.85%(61/65)的鼠疫菌具备4个毒力因子,96.97%(32/33)的鼠疫菌为强毒菌。结论青海省乌兰县分离的鼠疫菌具备青藏高原鼠疫病原体特性,鼠疫菌的毒力强,因此该地区的鼠疫防治工作不容松懈。     

黄连素作用鼠疫耶尔森菌的体外表达谱

目的 探讨黄连索抑制鼠疫耶尔森菌(简称鼠疫菌)作用的分子机制.方法 应用鼠疫菌全基因组芯片表达谱进行黄连素作用鼠疫菌研究.液体稀释法测定黄连索作用鼠疫菌的最小抑菌浓度(MIC).应用10倍MIC的黄连索作用于鼠疫菌30 min后,提取鼠疫菌总RNA,反转录合成eDNA,Cy3,Cy5荧光染料标记,芯片杂交,扫描仪扫描,SAM软件分析结果 .结果 在鼠疫菌全基因组范围内黄连素作用鼠疫菌的明显差异表达基因有360个.其中明显上调基因333个,下调基因27个.依据鼠疫菌CO92株基冈功能分类结果 ,这些差异基因分属24个不同的功能类群,其中主要有细胞膜相关基冈83个,未知功能基因75个,转运结合蛋白基因48个.发生明显改变的基因功能类群是代谢相关基因,共有40个.结论 获得了黄连素作用鼠疫菌的表达谱.阐述了黄连作用鼠疫菌的分子机制,主要机制在于影响代谢相关基因的上调.     

对1株疑似鼠疫耶尔森氏菌的系统鉴定

目的对1株可疑菌株进行系列验证实验,以确定其是否为鼠疫耶尔森氏菌（以下简称鼠疫菌）。方 法用鼠疫细菌学常规方法和分子生物学手段确定其生物学表型特征、特异性基因及基因组特征。结果该菌 株具备鼠疫菌的典型形态特征;能被鼠疫噬菌体完全裂解;主要生化特性为阿胶糖（＋）、鼠李糖（－） 、麦芽糖（＋）、蜜二糖（－）、甘油（＋）、脱氮（+）,与典型鼠疫菌一致。毒力因子检查结果为均 为阴性;对实验动物小白鼠完全无致死能力。全基因组芯片杂交实验和PCR扩增表明55023菌株没有鼠疫菌 的三个质粒;也不具鼠疫标识基因;pgm位点代表性基因YPO1954扩增阳性,YPO1908扩增阴性,表明其pgm 位点不完整;差异片段（DFR）分型结果表明该菌株缺失了14个DFR,不符合鼠疫菌的特征;多位点序列分 型（MLST）分析结果表明该菌株与鼠疫存在16个碱基的差异,而与血清III型假结核耶尔森氏菌仅相差两 个碱基。结论尽管55023菌株具备鼠疫菌的一些表型特征,但基因组特征表明其不是鼠疫菌,而可能是血 清III型的假结核耶尔森氏菌;噬菌体裂解结果等表型不能作为鼠疫菌鉴定的最终标准。     

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.     

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.     

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

Aim

Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5 mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors.

Methods

Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n = 3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction).

Results

At baseline, transgenic rats had +18 mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P < 0.05) without significant changes for the vehicle groups throughout the study (P > 0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P < 0.05) in transgenic and +25%, +5.3% and +6.7% (P > 0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P > 0.05).

Conclusion

Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.     

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one-week-and two-month-old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week-and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1% displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increases during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreacti vitv.     

Effects of pinealectomy and melatonin administration on certain indices of ovarian hyperplasia and/or hypertrophy in rats with both ovaries intact or after unilateral ovariectomy

Abstract: In earlier studies from other laboratories it was shown that melatonin decreased ovarian weight in rats and inhibited compensatory hypertrophy of the remaining ovary after unilateral ovariectomy. This study was designed to examine the influence of melatonin on certain indices of ovarian hyperplasia and/or hypertrophy in adult female rats with both ovaries preserved and with either an intact pineal gland or with the pineal gland removed (pinealectomy, PX) or, finally, in sham-PX animals. Similar studies were conducted on rats after unilateral ovariectomy, referring the examined parameters to the remaining intact ovary. The studies included mitotic activity of granulosa layer cells and corpus luteum cells, ovarian weight, ovarian cross-sectional area, cross-sectional area of the granulosa layer of all the Graafian follicles and the cross-sectional areas of the corpora lutea, visible on the ovarian cross-section. On the basis of results, we conclude that: 1) the effect of PX on the processes of ovarian hyperplasia and hypertrophy may vary; analogously, exogenous melatonin administration may influence ovarian hyperplasia and hypertrophy in different ways; 2) PX and exogenous melatonin may, under certain conditions, exert similar biological effects, even synergistic effects; 3) melatonin inhibits ovarian growth processes, while the effects of PX are variable; 4) the results indicate that in experiments performed on rats, with the use of two control groups, i.e., intact and sham-PX, melatonin effects on these two groups may differ.