Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cgamma signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.     

The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel

The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain-domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT's kinase activity (reduced by 55-75%) were identified. Mutations at Thr149 (37 ? distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT's kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.     

Validation of a chloroquine-induced cell death mechanism for clinical use against malaria

An alternative antimalarial pathway of an ‘outdated'' drug, chloroquine (CQ), may facilitate its return to the shrinking list of effective antimalarials. Conventionally, CQ is believed to interfere with hemozoin formation at nanomolar concentrations, but resistant parasites are able to efflux this drug from the digestive vacuole (DV). However, we show that the DV membrane of both resistant and sensitive laboratory and field parasites is compromised after exposure to micromolar concentrations of CQ, leading to an extrusion of DV proteases. Furthermore, only a short period of exposure is required to compromise the viability of late-stage parasites. To study the feasibility of this strategy, mice malaria models were used to demonstrate that high doses of CQ also triggered DV permeabilization in vivo and reduced reinvasion efficiency. We suggest that a time-release oral formulation of CQ may sustain elevated blood CQ levels sufficiently to clear even CQ-resistant parasites.Along with improvements in vector control, surveillance/diagnosis and treatment accessibility, the development of new drugs to counteract the problem of drug resistance remains integral to the eradication agenda.¹ Efforts to develop novel antimalarials have been promising,^{2, 3} and drugs designed specifically to reverse drug resistance are also being uncovered.⁴ However, novel chemical entities are expensive to test and take considerable time before they can be deployed. In comparison, alternative strategies to fully exploit the existing arsenal of antimalarials (largely already affordable and accessible) are likely to be relatively expedient and cost-effective.We had previously demonstrated the existence of a novel parasite programmed cell death (PCD) mechanism that was induced by high concentrations of chloroquine (CQ) and shown that clan CA cysteine proteases were key mediators of the pathway.⁵ We had also observed that the permeabilization of the parasite digestive vacuole (DV) was an important upstream trigger of this pathway and that other lysosomotropic compounds that are not parasite-specific could similarly destabilize the DV to initiate parasite PCD.⁶ We hypothesize that by altering the dosing regimen or formulation of CQ, it might be possible to reinstate CQ into antimalarial chemotherapy by making use of this novel mechanism.⁷In this present study, we begin by showing evidence that CQ treatment is able to result in the extrusion of DV proteases into the parasite cytoplasm. Second, we validate the existence of this PCD pathway in multiple laboratory strains and field isolates to suggest its clinical relevance and universality. Third, we investigate the minimum concentration and duration required for CQ to trigger PCD to determine if the pharmacokinetics of the current CQ regimen might be suitable for initiating PCD. Finally, we make use of two murine malaria models to demonstrate that a short exposure to high levels of CQ is able to induce parasite DV permeabilization in vivo and that this procedure reduces parasite viability.     

IL-31 Associated with Coronary Artery Lesion Formation in Kawasaki Disease

Background

Kawasaki disease (KD) is known to be associated with T help (Th) 2 reaction and subsequently allergic diseases. Interleukin-31 (IL-31) has also been reported to be involved in Th2 mediated diseases such as allergic diseases. However, the role of IL-31 in KD has not been previously reported. The aim of this study is to investigate whether IL-31 is associated with KD and its clinical outcome.

Material

A total of 78 KD patients who met the criteria of KD were enrolled in this study as well as 20 age-matched controls. Plasma samples were conducted to measure IL-31 before intravenous immunoglobulin (IVIG) treatment (KD1), within 3 days after IVIG treatment (KD2) and at least 3 weeks after IVIG treatment (KD3) by utilizing enzyme-linked immunosorbent assay (ELISA).

Result

Our findings showed that IL-31 expression was higher in KD patients after IVIG treatment significantly (KD2>KD1: 1265.0±199.3 vs. 840.2±152.5 pg/ml, p<0.0001). Further analysis revealed that IL-31 level was significantly higher in KD patients with coronary artery lesion (CAL) (656.6±139.5 vs. 1373.0±422.0 pg/ml, p = 0.04) before IVIG treatment (KD1). There were no significant differences between the IVIG resistance and IVIG responsiveness groups.

Conclusion

IL-31 was increased after IVIG treatment in patients with KD and was significantly associated with CAL formation. The results from this study may help to identify a novel risk factor for predicting KD and CAL formation.     

Laser Acupuncture Therapy in Patients with Treatment-Resistant Temporomandibular Disorders

Objective

To investigate the clinical effects of laser acupuncture therapy for temporomandibular disorders (TMD) after ineffective previous treatments.

Methods

A retrospective observational study was conducted in 29 treatment-resistant TMD patients (25 women, 4 men; age range, 17–67 years). Subjects were treated 3 times per week for 4 weeks with the Handylaser Trion (GaAlAs laser diode, 810 nm, 150 mW, pulsed waves), which delivered 0.375 J of energy (5 s) to ST7, ST6, and LI4 and 3 J (40 s) to each Ashi point, 7.5–26.25 J/cm² in total. The visual analog scale (VAS) and maximal mouth opening (MMO) were evaluated before and after treatment.

Results

VAS analysis showed that the patients were free of pain at rest (endpoint) after 5.90±6.08 sessions of laser acupuncture for acute TMD and after 16.21±17.98 sessions for chronic TMD. The VAS score on palpation of the temporomandibular joint reduced to 0.30±0.67 for patients with acute TMD (p = 0.005) and to 0.47±0.84 for those with chronic TMD (p<0.001). The MMO significantly increased in patients with acute TMD (7.80±5.43 mm, p = 0.008) and in patients with chronic TMD (15.58±7.87 mm, p<0.001).

Conclusions

Our study shows that laser acupuncture therapy improves the symptoms of treatment-resistant TMD. Further studies with a more appropriate design, involving long-term follow-up examinations in a larger patient sample, are needed to evaluate its efficacy.     

CFS-1686 Causes Cell Cycle Arrest at Intra-S Phase by Interference of Interaction of Topoisomerase 1 with DNA

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin''s induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.     

RNA干扰应用新进展

RNAi是dsRNA诱导的序列特异的基因沉默,siRNA是主要诱因。该文重点综述siRNA的特征、合成方式、转染及转染后检测方法的研究进展;RNAi技术在细胞信号传导途径分析、冗余基因确定、基因功能检测、基因治疗、药物开发等研究领域应用新进展及在相关应用领域仍待解决的问题。     

系统发生分析发现牛病毒性腹泻病病毒新基因亚型

本研究对我国首次分离获得的牛源牛病毒性腹泻病毒(BVDV)毒株Changchun 184(CC-184)和猪源牛病毒性腹泻病毒ZM-95进行了遗传衍化关系研究.选择主要抗原E2基因为研究对象,首先应用RT-PCR及套式PCR克隆得到CC-184和ZM-95的E2片段,通过序列测定发现CC-184和ZM-95 E2基因长度分别为1,122bp和1,125bp,各自编码374和375个氨基酸残基.核酸序列同源性比较和系统发生分析表明2株病毒均属于BVDV-1,CC-184与Osloss亲缘关系最近,都属于已有的b基因亚型,其E2基因同源性达91.8%.而ZM-95的E2基因有一个特征性的变异区,包含一个密码子序列插入,这一变异区编码了一段有别于其他瘟病毒的五肽氨基酸序列HYKKK.结果还表明ZM-95与BVDV-1现有的5个基因亚型的亲缘关系均较远,E2基因同源性最高(与Oregonc24v)只有72.4%.而BVDV 1亚型内毒株间的同源性大于85%,亚型间的同源性在69%～75%之间,充分说明ZM-95是BVDV-1中一个新发现的基因亚型.通常认为猪源BVDV来源于牛,应该与牛源BVDV有十分近的遗传关系,但是本研究发现ZM-95与其他已知牛源BVDV较低的基因同源性说明猪源BVDV还具有独立的遗传衍化与传播来源的可能性.     

巴斯德毕赤酵母表达的突变型人白细胞介素-2的发酵条件与纯化研究

在以前的研究中,通过蛋白质工程技术获得了三突变体白细胞介素_2基因(编码125位半胱氨酸→丙氨酸;18位亮氨酸→蛋氨酸;19位亮氨酸→丝氨酸) ,并在毕赤酵母中加以表达。进一步优化表达条件,其最适诱导条件:80 %以上的通气,诱导2d ,初始pH60 ,甲醇终浓度为10%。在上述条件下表达量占菌体总蛋白的30%以上,大约200mg L。建立了一套从毕赤酵母表达上清中分离纯化分泌型表达蛋白IL_2的方法,经离心,超滤浓缩,强阳离子交换S柱和分子筛层析得到纯化的突变型和野生型IL-2 ;其得率为27% ,纯度达电泳纯并且HPLC检测只有一个峰。纯化的突变蛋白对CTLL-2细胞具有刺激性;与野生型IL-2相比,在各种温度条件下储存的突变蛋白保留有更高的活性;突变型IL-2的活力是野生型的4～5倍,具有更高的利用价值。