蝎形引物PCR快速检测环境水样中沙门菌方法研究

[目的]以蝎形引物PCR技术为基础建立一套环境水样沙门菌的快速检测方法。[方法]选择沙门菌himA基因设计出以荧光素(FAM)和淬灭物(DABCYL)双标记探针的茎环尾状结构构成蝎形引物，对8种沙门菌和8种非沙门菌经增菌、DNA提取后，以蝎形引物PCR方法进行检测，探讨该检测方法的可行性、特异性和灵敏度，并与普通引物PCR法检测50份环境水样进行比较。[结果]蝎形引物PCR方法检测沙门菌，细菌密度与荧光相对强度存在着量比关系，与普通引物PCR法比较，具有快速、特异、灵敏度高的特点，在50μl PCR及反应体系中最低可检出2cfu沙门菌。[结论]沙门菌蝎形引物在PCR扩增中，探针可特异地与目的DNA片段杂交，使DABCYL不能有效地淬灭FAM产生的荧光，荧光可经仪器直读检出。所建立的快速检测方法可定性或定量的用于环境水样中沙门菌检测。     

Differential involvement of disulfide bridges on the folding of a scorpion toxin

Leiurotoxin I is a neurotoxin, blocker of Ca²⁺-activated apamin-sensitive K⁺ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys³-Cys²¹, Cys⁸-Cys²⁶ and Cys¹²-Cys²⁸. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were replaced by α-aminobutyrate residues to suppress one disulfide bridge, were analyzed by ¹H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys³-Cys²¹ and a loss of organized structure for another analog lacking disulfide bridge Cys¹²-Cys²⁸. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys¹²-Cys²⁸ is essential for the folding process. In contrast, the lack of disulfide bridge Cys³-Cys²¹ does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.     

Intra-arterial injection of Mesobuthus tamulus venom elicits cardiorespiratory reflexes involving perivascular afferents.

Role of perivascular afferents for the cardiorespiratory alterations produced by Mesobuthus tamulus (BT) envenomation was examined in urethane-anaesthetized male rats. Blood pressure (BP), respiratory rate (RR) and heart rate (HR) were recorded after injecting BT venom/saline in the distal end of femoral artery for 60 min. In addition, paw oedema was also determined. Injection of venom produced an immediate (within 2 s) increase in RR followed by a decrease and finally a sustained increase up to 60 min. BP was increased (within 10 s) by 30-50%, which gradually declined but remained above the initial level up to 60 min. The bradycardiac response was late to occur (after 50 s) and the peak response was seen between 10 and 50 min, which remained at that level. There was oedema in the ipsilateral hind paw (venom injected side) as compared to contralateral side and saline control group. The oedema and cardiorespiratory changes were maximal at 1.0 mg/kg of venom. Pretreatment with indomethacin significantly attenuated the venom-induced responses and also blocked the paw oedema. Present experiments reveal that BT venom in a segment of an artery produces oedema by involving prostaglandins to sensitize the nociceptors present in perivascular tissues to evoke the cardiorespiratory reflexes.     

Immunosuppressive evidence of Tityus serrulatus toxins Ts6 and Ts15: insights of a novel K+ channel pattern in T cells

The voltage‐gated potassium channel Kv1.3 is a novel target for immunomodulation of autoreactive effector memory T cells, which play a major role in the pathogenesis of autoimmune diseases. In this study, the Ts6 and Ts15 toxins isolated from Tityus serrulatus (Ts) were investigated for their immunosuppressant roles on CD4⁺ cell subsets: naive, effector (T_EF), central memory (T_CM) and effector memory (T_EM). The electrophysiological assays confirmed that both toxins were able to block Kv1.3 channels. Interestingly, an extended Kv channel screening shows that Ts15 blocks Kv2.1 channels. Ts6 and Ts15 significantly inhibit the proliferation of T_EM cells and interferon‐γ production; however, Ts15 also inhibits other CD4⁺ cell subsets (naive, T_EF and T_CM). Based on the Ts15 inhibitory effect of proliferation of all CD4⁺ cell subsets, and based on its blocking effect on Kv2.1, we investigated the Kv2.1 expression in T cells. The assays showed that CD4⁺ and CD8⁺ cells express the Kv2.1 channels mainly extracellularly with T_CM cells expressing the highest number of Kv2.1 channels. We also provide in vivo experimental evidence to the protective effect of Ts6 and Ts15 on delayed‐type hypersensitivity reaction. Altogether, this study presents the immunosuppressive behaviour of Ts6 and Ts15 toxins, indicating that these toxins could be promising candidates for autoimmune disease therapy. Moreover, this is the first report illustrating the involvement of a novel K⁺ channel subtype, Kv2.1, and its distribution in T‐cell subsets.     

Fatal ischemic stroke following Tityus serrulatus scorpion sting in a patient with essential thrombocythemia

Context: Stroke following scorpion stings is rare. We report a fatal envenomation involving multiple, extensive brain infarcts in a patient with a previous diagnosis of essential thrombocythemia (ET) who was stung by Tityus serrulatus (T. serrulatus).

Case details: A 44-year-old woman with a diagnosis of low-risk ET (platelets <1,000,000/mm³, age <60 years and no history of thrombosis; positive JAK2V617F mutation) was admitted to a local ED 1?h after being stung by T. serrulatus on the left foot. She developed signs of severe envenomation, including several episodes of profuse vomiting, pallor and confusion soon after the sting, followed by shock (BP: 90/60 to 60/40?mmHg) and was treated with scorpion antivenom, vasopressors and mechanical ventilation. A brain computed tomography (CT) scan (54-h poststing) revealed diffuse bilateral cerebellar hypodensity, with partial involvement of both occipital lobes and thalamus, obstructive hydrocephaly with signs of cerebrospinal fluid extravasation, and ascending transtentorial herniation, suggestive of bilateral ischemia involving the posterior cerebral circulation. External ventricular drainage resulted in no improvement and brain death was confirmed on day 10.

Discussion: Several mechanisms have been proposed to explain stroke following scorpion stings, such as sympathetic stimulation, myocardial dysfunction, hypotension/shock, arrhythmias and coagulopathy. Ischemic stroke is one of the most serious complications of ET. The risk factors for thrombotic/ischemic events in patients with ET include age (≥60 years) and previous vascular events. Severe scorpion envenomation resulting in myocardial dysfunction and systemic inflammatory response syndrome may increase the overall risk of arterial thrombosis in this patient.     

三种全蝎粉水提液不同活性部位的蛋白质含量测定及其抗肿瘤活性研究

目的：比较三种全蝎粉水提液各活性部位的蛋白质含量及其对肺癌细胞的抑制作用。方法：野生全蝎粉经过仿胃肠道的酶解液,发酵全蝎粉、野生全蝎粉的水提溶液,分别超滤、冻干,浓缩液用MTT法测定对肺癌细胞的抑制率。结果：三种全蝎粉水提液各活性部位对肺腺癌细胞的抑制率与空白对比差异有统计学意义（P〈0.05,P〈0.01）。结论：不同方法处理的全蝎粉各活性部位均对肺癌细胞有抑制作用,且多为低浓度抑制率较高,小分子寡肽对肺癌细胞的抑制作用最好。     

Iron Ingestions—The Children's Mercy Hospital

Abstract

Scorpion envenomation is a common medical problem and life hazard in many countries of the world. Scientific investigations have addressed the interrelationship between the stimulatory effects of the venom on the autonomic nervous system and adrenals and the subsequent effects of released transmitters on the cardiovascular system. A number of clinical cardiovascular syndromes may dominate the initial clinical presentation after envenomation: the syndromes usually vary with the age of the victim, the size of the offender and the season. Central nervous system dysfunction is seen in children but rarely observed in adults; if accompanied by severe hypertension the clinical picture is consistent with acute hypertensive encephalopathy. Heart failure, pulmonary edema or a shock-like syndrome has been observed in 25% and hypertension in 30% to 77% of our patients. The electrocardiographic abnormalities recorded in the majority of the patients after envenomation include an “acute myocardial infarction-like pattern.” Rhythm disturbances are frequent but conduction abnormalities are rare. Echocardiographic, radionuclide and experimental hemodynamic observations have provided evidence that heart failure and pulmonary edema after envenomation are multifactorial with diminished systolic performance following the initially increased left ventricular contractility and decreased ventricular diastolic compliance. Clinical laboratory data reporting increased catecholamine metabolite excretion and elevated plasma renin and aldosterone are consistent with the stimulatory effects of the venom on the autonomic nervous system. Treatment, including our experience with vasodilators and calcium channel blockers, is reviewed.     

蝎毒多肽增强5-氟尿嘧啶对H22肝癌抑制作用的机制研究

目的 探讨蝎毒多肽增强5-氟尿嘧啶（5-Fu）对肝癌H₂₂抑制作用的机制。方法 以接种H₂₂肝癌小鼠腹水方法建立小鼠荷瘤模型,在接种后第7天将荷瘤小鼠随机分为模型组、蝎毒多肽（20 mg/kg）组、5-Fu（15 mg/kg）组、低剂量蝎毒多肽（5 mg/kg）＋5-Fu组、高剂量蝎毒多肽（20 mg/kg）＋5-Fu组,每组10只,连续给药21 d。绘制肿瘤体积增长曲线并计算抑瘤率;CD34标记肿瘤微血管;免疫组织化学法检测核因子-κB（NF-κB）、基质金属蛋白酶-9（MMP-9）和血管内皮生长因子（VEGF）蛋白表达。结果 与模型组相比,联合给药组H₂₂肝癌移植瘤的生长受到明显抑制（P＜0.01）,微血管密度（MVD）及NF-κB、MMP-9、VEGF蛋白表达均明显降低（P＜0.01）;与5-Fu组相比,高剂量蝎毒多肽＋5-Fu组的MVD及NF-κB、MMP-9、VEGF蛋白表达也显著降低（P＜0.01）,而低剂量蝎毒多肽＋5-Fu组则无显著改变。结论 蝎毒多肽可增加5-Fu对肿瘤组织的抑制作用,其机制可能与抑制H₂₂肝癌组织NF-κB通路及MMP-9、VEGF的表达,从而抑制新生血管形成有关。     

蝎毒组分Ⅲ对4种白血病细胞增殖的影响

目的 探讨蝎毒组分Ⅲ(SVC-Ⅲ)对4种白血病细胞增殖及细胞周期的影响.方法 将常规培养的白血病细胞接种子细胞培养板上,分为正常对照组和不同浓度的SVC- Ⅲ刺激组,MTT检测细胞的增殖,流式细胞术检测细胞周期分布.结果 浓度＞1 mg/L SVC-Ⅲ刺激对白血病细胞的增殖具有剂量效应和时间效应的依赖关系,同时细胞周期被阻滞在G0/G1期,与正常对照组相比差异具有显著性(P＜0.01或0.05).结论 SVC-Ⅲ可能通过细胞周期的调控而抑制细胞的增殖.     

蝎毒多肽提取物对K562细胞PI3K和p-Akt信号蛋白表达及细胞增殖的影响

本研究旨在探讨蝎毒多肽提取物(PESV)对人慢性髓系白血病细胞系K562细胞凋亡调控的PI3K和p-Akt信号蛋白的影响及作用机制。将体外培养的K562细胞,经PESV处理不同时间后,通过MTT法检测细胞增殖曲线,Western blot法检测PI3K及p-Akt蛋白水平变化。结果表明,与对照组相比,PESV处理后K562细胞凋亡率增加,PI3K及p-Akt蛋白表达降低。结论:PESV可能通过抑制凋亡调控的PI3K、p-Akt信号蛋白,抑制K562细胞增殖,促进细胞凋亡。