Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages

Background and purpose:

Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.

Experimental approach:

Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.

Key results:

Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-κB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.

Conclusions and implications:

Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.     

Prevalence of Murine Helicobacter spp. Infection Is Reduced by Restocking Research Colonies with Helicobacter-Free Mice

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.Abbreviation: EHS, enterohepatic Helicobacter spp.; ET, embryo transfer; Hb, H. bilis; Hh, H. hepaticus; Hm, H. mastomyrinus; Hr, H. rodentium; Ht, H. typhlonicusEnterohepatic Helicobacter spp. (EHS) infections are endemic in the majority of research mouse colonies. In 2007, 84% of mice shipped from academic institutions worldwide for embryo transfer (ET) rederivation at our institution were PCR-positive for EHS. H. hepaticus (Hh) was detected in 64% of the mouse shipments either as a monoinfection or in combination with other EHS including H. bilis (Hb), H. rodentium (Hr), H. typhlonicus (Ht), and H. mastomyrinus (Hm).³⁰ Although EHS generally cause subclinical infection in immunocompetent mice, opportunistic infections have the potential to confound experimental data in mouse models.^9,17,34 Importantly, chronic EHS infection in immunodeficient and select inbred strains of mice can induce liver¹⁰ and lower bowel carcinoma,¹³ typhlocolitis, and rectal prolapse,^16,21,28 and reduce reproductive performance.²⁵ In addition, EHS-induced inflammatory responses may alter host immune responses to unrelated experimental infections (for example, promoting elevated systemic IFNγ responses).^3,20Key challenges to eradication of EHS from rodent colonies are determining infection status, eliminating endemic infections, and instituting management practices that prevent reinfection. EHS are disseminated through fecal–oral transmission within a colony and are transmissible to surveillance mice through dirty-bedding exposure.^1,19,24,32 For routine surveillance, PCR assay of feces or cecal mucosal scrapings for genus-specific Helicobacter 16S rRNA genes is the most efficient means of detecting EHS infection, with speciation (if desired) of positive results by culture, restriction fragment length polymorphism analysis, species-specific PCR, or sequence analysis.³⁴ In 1999, as determined by species-specific PCR assays of cecal scrapings from 59 surveillance mice exposed to dirty bedding from colony mice in 26 rooms representing 4 mouse facilities, EHS were endemic on our campus, with prevalence in surveillance mice of 41% for Hh, 82% for Hr, and 6% for Hb.³² Husbandry practices used to minimize cage-to-cage transmission of EHS included microisolation caging, sanitized forceps to transfer mice, and a cage change order from known Helicobacter-free mice to mice of unknown or known EHS infection status (that is, clean to dirty traffic flow of personnel and equipment).³² Although EHS eradication potentially could be accomplished campus-wide by using labor-intensive antibiotics^7,15 and cross-fostering,^4,29,31 we hypothesized that a more cost-effective approach, without confounding experimental data, would be to restrict importation of mice to EHS-free sources. Vendors were screened to establish that production colonies were SPF for EHS, and a new requirement was instituted for embryo transfer (ET) rederivation of mice obtained from random sources, typically other academic institutions, replacing traditional quarantine practices. This study used PCR data from 1999 and 2009 to evaluate the success of this approach, which was defined as a marked decrease in the prevalence of EHS infection over time.     

Catheter ablation of accessory pathways, atrioventricular nodal reentrant tachycardia, and the atrioventricular junction: final results of a prospective, multicenter clinical trial. The Atakr Multicenter Investigators Group

BACKGROUND: The purpose of this study was to evaluate the safety and efficacy of a temperature-controlled radiofrequency catheter ablation system. METHODS AND RESULTS: The patient population included 1050 patients who had undergone ablation of atrioventricular nodal reentrant tachycardia (AVNRT), an accessory pathway (AP), or the atrioventricular junction (AVJ). Ablation was successful in 996 patients. The probability of success was highest among patients who had undergone ablation of the AVJ, lowest in patients who had undergone ablation of an AP, and in between for patients who had undergone ablation of AVNRT. A major complication occurred in 32 patients. Four variables predicted ablation success (AVJ, AVNRT, or left free wall AP ablation and an experienced center). Four factors predicted arrhythmia recurrence (right free wall, posteroseptal, septal, and multiple APs). Two variables predicted development of a complication (structural heart disease and the presence of multiple targets), and 3 variables predicted an increased risk of death (heart disease, lower ejection fraction, and AVJ ablation). CONCLUSIONS: These findings may serve as a guide to clinicians considering therapeutic options in patients who are candidates for ablation.     

中药材龟板和鳖甲中DNA的提取与扩增

中药材龟板和鳖甲中ＤＮＡ的提取与扩增王亚明，周开亚，吴平，徐珞珊（南京师范大学生物系，南京２１００９７；中国药科大学生药学研究室，南京２１０００９）龟鳖类药材在我国使用历史悠久，具补阴益气的功效。《中国药典》（１９９０年版）规定龟甲（龟板）的原动物为...     

Expression and characterization of Gag protein of HIV-1（CN） in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strain （HIV-1 （CN）） in Pichia pastoris, the fulllength gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by Sail was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA （1.3 kb） was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kD, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13 % of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows： BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85 %. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.     

骨髓间充质干细胞移植后脑梗死大鼠脑电图的变化

目的:对比观察骨髓间充质干细胞移植前后脑梗死大鼠脑电图的变化。方法:实验于2002-09/12在解放军第三军医大学中心实验室及西南医院神经内科肌电图室完成。①实验分组:选取清洁级健康成年Wistar大鼠15只,随机数字表法分为干细胞移植组、模型对照组、假手术组,5只/组。②实验方法:另取2只健康幼年Wistar大鼠用于骨髓间充质干细胞的提取,联合采用密度梯度离心及贴壁法分离培养大鼠骨髓间充质干细胞,选取生长良好的1～3代细胞用于移植实验。干细胞移植组、模型对照组大鼠建立大脑中动脉栓塞模型。假手术组仅分离颈总动脉、颈外动脉和颈内动脉,不予结扎和放置线栓。造模后1周,干细胞移植组、假手术组大鼠行细胞移植,在立体定向仪定位下于脑梗死区(壳核)直接注射骨髓间充质干细胞悬液5μL,细胞浓度1×104μL-1,移植坐标为前囟前1.0mm,右旁开3.0mm,硬膜下5.0mm。模型对照组大鼠于相同部位注射等量不含细胞的磷酸盐缓冲液。③实验评估:采用脑电图机分别于造模前、造模后1周(移植前)、细胞移植后4周对各组大鼠进行脑电图检测。结果:15只大鼠均进入结果分析。①造模前基本节律为8～11Hz、15～30μV的α波,间或少量θ波,双侧对称。②造模后1周,假手术组异常率为0;模型对照组20%(1/5)轻度异常,80%(4/5)中度异常;干细胞移植组20%(1/5)轻度异常,60%(3/5)中度异常,20%(1/5)重度异常。③细胞移植后4周,假手术组脑电图恢复正常;模型对照组随术后时间的延长慢波有所减少,但仍可见到δ波、棘波、棘慢波的发放,至细胞移植后4周60%(3/5)轻度异常,40%(2/5)中度异常;干细胞移植组术后局限性慢波逐渐减少,基本节律全部恢复为α波,不对称的情况明显好转,至细胞移植后4周60%(3/5)轻度异常,以病灶侧局限性θ波较多为主,另外40%(2/5)基本正常。结论:动物实验显示骨髓间充质干细胞移植对脑梗死大鼠的脑电图背景节律有改善作用,一定程度上促进了神经系统功能的恢复。     

脊髓损伤后真皮多能干细胞移植时机的选择

目的:观察脊髓损伤后不同时间移植真皮多能干细胞对大鼠运动功能修复的影响。方法:实验于2006-06/2007-03在第三军医大学生理学教研室完成。①实验材料:实验动物2～4月龄SPF级SD大鼠,体质量(210±40)g,雌雄不限,由第三军医大学实验动物中心提供。真皮多能干细胞为第三军医大学防原医学系从SD大鼠真皮中提取和分离。②实验方法:将42只SD大鼠在L4水平制成脊髓全横断损伤模型。将动物随机分为对照组(n=6)、真皮多能干细胞移植组(n=36)。真皮多能干细胞移植组又分为6个时间点:损伤后1,4,7,10,14,21d移植组,每组6只。各移植组于伤处移植大鼠真皮多能干细胞,而对照组于损伤后7d注射等量磷酸盐缓冲液。③实验评估:分别于移植后1d、1周、4周、8周、12周对各组大鼠进行动物行为学和脊髓诱发电位检测。结果:42只实验大鼠均进入结果分析,无脱落。①动物行为学评分:4周以后各组动物行为学评分比较差异有显著性意义(P<0.05),移植组明显高于对照组(P<0.05),各移植组间比较,以损伤后7,10d移植组动物行为学评分改善最显著。②各组大鼠脊髓诱发电位检查:体感诱发电位和运动诱发电位潜伏期和波幅值于移植后8,12周后明显高于对照组(P<0.05),各移植组间比较,以损伤后7,10d移植组体感诱发电位和运动诱发电位潜伏期和波幅值改善最显著。结论:脊髓损伤后7～14d进行真皮多能干细胞移植可明显改善大鼠后肢的运动功能。     

Older peoples' perceptions of oral health: 'it's just not that simple'

Abstract: Objectives: Little is known about older persons’ perceptions of oral health and oral health care. The purpose of this study was to explore the viewpoint of older adults’ regarding their oral health care practices. Methods: A qualitative interpretive methodology was employed comprising three analytic levels: coding of data into concepts, analysis of concepts into themes, followed by an in‐depth analysis of relationships within concepts and between themes. In‐depth individual interviews were conducted with 19 participants aged 65 to 87 years. Results: Older people’s decision to access oral health care involves complex and personally meaningful strategies. A dental visit surfaces hopes and fears based on past and present experiences. Mouth and teeth are not merely objects of dental care; they represent a person’s social and relational self. Age‐related changes challenge the relational self as represented in societal ideal images of youth and perfection (the perfect smile). This study highlights older peoples’ resilience and determination when faced with the dilemmas in accessing oral health care – it costs, personally as well as financially. Contrary to the assumption that older peoples’ oral health status is related to neglect, rather for many, it is the result of the intersection of their history with technological advances. Conclusions: These findings challenge oral health care practitioners to be sensitive to the contexts affecting their older client’s oral health care status. They do not ‘just go’ to the dentist; they bring with them their past dental experiences and their hopes for the future. It matters how one is treated at this vulnerable time.