TLR4在宫颈癌与不同HPV亚型宫颈癌细胞株中表达及意义

目的 探讨TLR4在正常宫颈组织、宫颈上皮不典型增生、宫颈癌组织以及不同HPV亚型宫颈癌细胞株中表达与意义.方法 采用SP免疫组织化（IHC）检测TLR4在12例正常宫颈组织、30例宫颈轻度不典型增生（cervical intraepithelial neoplasia Ⅰ,CINⅠ）、30例宫颈中-重度不典型增生（cervical intraepithelial neoplasiaⅡ-Ⅲ,CIN Ⅱ-Ⅲ）以及49例宫颈癌（invasive cervical carcinoma,ICC）组织中的表达;应用细胞免疫荧光法、逆转录聚合酶链反应（RT-PCR）检测TLR4在不同HPV亚型宫颈癌细胞株HPV18（＋）HeLa、HPV16（＋）Siha以及HPV（-）C33a宫颈癌细胞株上的表达.结果 TLR4的表达随着宫颈病变程度的增高逐渐增强,在正常宫颈组织、CINⅠ、CIN Ⅱ～Ⅲ、ICC中阳性表达率分别为8.33 %（1/12）、20.00 %（6/30）、26.67 %（8/30）和55.10 %（27/49）,ICC与正常宫颈组织、CINⅠ、CINⅡ～Ⅲ之间均有显著性差异（P＜0.01）.TLR4表达与HPV感染有关,在HPV阳性宫颈癌细胞株上表达强于HPV阴性细胞株.结论 TLR4可能参与宫颈癌起始、发展,TLR4的表达与功能与HPV感染有关.     

Expression and Functional Analysis of Toll-like Receptor 4 in Human Cervical Carcinoma

Toll-like receptors are expressed in human immune cells and many tumors, but the role of toll-like receptor 4 (TLR4) in the development of tumors is controversial. We demonstrated the expression, distribution, and functional activity of TLR4 in tissues of normal cervix, cervical intraepithelial neoplasia (CIN), invasion cervical cancers (ICC), and different human papillomavirus (HPV)-infected cervical cancer cells. The results showed that TLR4 expression was in accordance with the histopathological grade: higher in ICC than in CIN, and low in normal cervical tissues and malignant cervical stroma. Expression was higher in SiHa (HPV16+) than in HeLa (HPV18+) cells, but was not observed in C33A (HPV?) cells. After treatment with its agonist, lipopolysaccharide (LPS), the expression levels of TLR4 was increased and apoptosis resistance was induced in SiHa cells, but not in HeLa or C33A cells. Meanwhile, LPS treatment did not alter the cell cycle distribution in SiHa cells. The mechanism of apoptosis resistance may be related to HPV16 infection and not correlated with the cell cycle distribution. Targeting TLR4 in combination with traditional drug treatment may serve as a novel strategy for more effectively killing cancer cells.     

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4′ positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-l-phenylalanine residues at 4 and 4′ positions. Binding values are: K_\texti^m = 0.51 \textnM K_{\text{i}}^{\mu } = 0.51\,{\text{nM}} and K_\texti^d = 12.8 \textnM K_{\text{i}}^{\delta } = 12.8\,{\text{nM}} for compound 9, K_\texti^m = 0.09 \textnM K_{\text{i}}^{\mu } = 0.09\,{\text{nM}} and K_\texti^d = 0.11 \textnM K_{\text{i}}^{\delta } = 0.11\,{\text{nM}} for analogue 10.     

Up-regulation of Foxp3 participates in progression of cervical cancer

Foxp3 was identified as a key protein in mediating inhibitory functions of regulatory T cell (Treg). Foxp3 was thought to express only in the T cell lineage until recently when some researches reported that Foxp3 was also expressed by cancer cells. In this study, we describe for the first time the expression of Foxp3 in cervical cancer. Progression from cervical intraepithelial neoplasia (CIN) to cervical cancer is a multistep process initiated by persistent infection with high-risk human papillomavirus (HPV). P16^INK4a is a crucial marker of HPV integration into host cells. In the present study, expressions of Foxp3 and P16^INK4a in CIN and cervical cancer were detected by immunohistochemistry. Our results found expression level of Foxp3 was increased during the progression of cervical neoplasia. Moreover, up-regulation of Foxp3 appeared to be correlated with the expression of P16^INK4a. Examination of the role of Foxp3 in differentiation by double immunostaining for cytokeratin 10 (CK10) showed significant association between Foxp3 expression and differentiation (Foxp3 vs CK10). Furthermore, positive expression of Foxp3 was correlated with tumor size. These data suggest that Foxp3 may play an important role in differentiation and growth of cervical cancer cells. Our findings provide new insights regarding the role of Foxp3 in differentiation and its association with HPV infection during the development of cervical cancer.     

Expression of the HPV18E2 Gene in Cervical Cancer and Premalignant Lesions and its Clinical Significance

This study aims to observe the expression of the HPV18E2 gene in cervical cancer and premalignant lesions and to investigate its clinical significance. The expression of the HPV18E2 gene in the cervical tissues obtained from 38 women with cervical lesions was detected using the RT-PCR method. The pathological changes were graded based on cervical intraepithelial neoplasia (CIN) criteria. The HPV18E2 gene was expressed mainly in cervical premalignant lesions, 60 % in Grade I CIN, 33.3 % in Grade II CIN, and 28.6 % in Grade III CIN. No expression was detected in cervical cancer and chronic cervical inflammation. This study suggests that peptides vaccine targeting the HPV18E2 protein may disrupt and prohibit the progress of diseases induced by HPV 18 infection (i.e., CIN and cervical cancer).     

Influence of {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ( \textM₇^\textIIMTs {\text{M}}_7^{\text{IIMTs}} ) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd₃-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd₃-thiolate cluster and the enfolding protein domain. Alterations in the \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological function.     

Expression of integrins and Toll-like receptors in cervical cancer: effect of infectious agents

We hypothesized that development of cervical cancer is associated with alterations in the expression of innate immune receptors, i.e. integrins and TLRs, and that these alterations can be induced by infectious agents. We have studied the expression of these proteins in cervical biopsy tissues and cervical cancer-derived cell lines HeLa, CaSki, SiHa, C-33?A, and ME180. Immunohistochemistry analysis demonstrated an increase in integrin αv, β3, β4, and β6 expression in the epithelium during the development of cervical cancer. A clear trend towards higher expression of integrin β6 in cell lines harbouring human papillomavirus (HPV) genetic material, compared to HPV-negative C-33?A, was observed. To investigate whether bacterial infection can alter the expression of TLRs and integrins, we infected HeLa cells by two pathogens, Escherichia coli and Pseudomonas aeruginosa, using a common bacterium of the female genital tract, Lactobacillus reuteri, as a control. Infection with E. coli or P. aeruginosa, but not with L. reuteri, significantly altered the expression of TLR and integrins, particularly of TLR4 and integrin β6. Considering that both integrin β6 and TLR4 play important roles in tumorigenesis, our data suggest that bacterial infection may trigger cancer development in HPV-infected cervical epithelium.     

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO₂ and high CO₂ conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO₂ conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and \textP_\textm^\textchla {\text{P}}_{\text{m}}^{{\text{chl}}a} , \textR_\textd^\textchla {\text{R}}_{\text{d}}^{{\text{chl}}a} , α^chla than under low CO₂ conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.     

Parameterization of chlorophyll a-specific absorption coefficients and effects of their variations in a highly eutrophic lake: a case study at Lake Kasumigaura, Japan

The chlorophyll a-specific absorption coefficient ( a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) ) in a highly eutrophic lake can show characteristics distinct from that in the ocean due to the differences in the structure and composition of phytoplankton. In this study, investigated the variation of a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) in Lake Kasumigaura, a highly eutrophic lake in Japan, in association with the package effect and the effect of accessory pigments, and carried out the parameterization of a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) . Although a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) did not vary spatially, it did show significant temporal variation, with a particularly high value after spring-bloom. This high a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) in spring was attributed to a lower package effect and a higher proportion of carotenoid than the other samples. Although the value of a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) was correlated with the concentration of chlorophyll-a (Chl-a), the correlation coefficient was lower than those reported in the ocean. Some lake-water samples showed variations of the package effect and the effect of accessory pigments that were independent of the concentration of Chl-a, and these independent variations resulted in the weak correlation between a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) and the concentration of Chl-a. Together, these results suggest that the factors controlling a_\textph^* ( l) a_{\text{ph}}^{*} \left( \lambda \right) in highly eutrophic lakes are distinct from that in ocean samples.     

宫颈癌中端粒酶的表达和高危型人乳头瘤病毒感染

目的:研究不同程度子宫颈病变中高危型人乳头瘤病毒HR-HPV感染和端粒酶活性的表达,以探讨两者在宫颈癌及宫颈上皮内瘤变中的作用及相关性。方法:采用第二代杂交捕获技术检测宫颈脱落细胞HPV-DNA含量,并用免疫组织化学EnVision二步法检测宫颈组织标本中端粒酶的表达。结果:(1)端粒酶阳性表达率在对照组、CINⅠ、CINⅡ、CINⅢ和宫颈癌组分别为10.00%、16.67%、40.00%、70.00%、95.00%,宫颈癌组高于CINⅢ,CINⅢ高于CINⅡ,CINⅡ高于CINⅠ,差异均有统计学意义(x2=4.329,P=0.037;x2=4.327,P=0.038;x2=4.022,P=0.045)。(2)随着宫颈病变级别的增加,高危型HPV的阳性率和病毒负荷量均增高。高危型HPV的阳性率在宫颈癌和CINⅢ组明显高于对照组、CINⅠ及CINⅡ(x2=29.501~7.414,P<0.01)。高危型HPV的病毒负荷量在对照组与其他4组比较,差异均有统计学意义(P<0.05);CINⅠ组分别与CINⅡ、CINⅢ及宫颈癌组比较差异均有统计学意义(P<0.05)。(3)随着宫颈病变级别的增加,高危型HPV的阳性率和端粒酶阳性表达率依次递增,两者有明显的相关性(r=0.943,P<0.01)。结论:高危型HPV感染和端粒酶活性均与宫颈癌前病变及宫颈癌的发生发展密切相关,有望作为子宫颈癌前病变和宫颈癌筛查的监测指标。     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Analysis of the activation mechanism of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase through an electron transfer chain

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an \textE_\textr \textC_\texti^￠ {\text{E}}_{\text{r}} {\text{C}}_{\text{i}}^{\prime } catalytic mechanism. An intermolecular electron transfer rate constant of (4 ± 1) × 10⁵ M⁻¹ s⁻¹ was estimated for both forms of the enzyme, as well as a similar Michaelis–Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

3D-QSAR studies of triazolopyrimidine derivatives of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydroorotate dehydrogenase inhibitors using a combination of molecular dynamics,docking, and genetic algorithm-based methods

A series of 35 triazolopyrimidine analogues reported as Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors were optimized using quantum mechanics methods, and their binding conformations were studied by docking and 3D quantitative structure–activity relationship studies. Genetic algorithm-based criteria was adopted for selection of training and test sets while maintaining structural diversity of training and test sets, which is also very crucial for model development and validation. Both the comparative molecular field analyses ( q_\textLOO² = 0.841 q_{\text{LOO}}^2 = 0.{841} , r_\textncv² = 0.99 r_{\text{ncv}}^2 = 0.{99} ) and comparative molecular similarity indices analyses ( q_\textLOO² = 0.757 q_{\text{LOO}}^2 = 0.{757} , r_\textncv² = 0.943 r_{\text{ncv}}^2 = 0.{943} ) show excellent correlation and high predictive power. Furthermore, molecular dynamics simulations were performed to explore the binding mode of the two of the most active compounds of the series, 10 and 14. Harmonization in the two simulation results validate the analysis and therefore applicability of docking parameters based on crystallographic conformation of compound 14 bound to receptor molecule. This work provides useful information about the inhibition mechanism of this class of molecules and will assist in the design of more potent inhibitors of PfDHODH.     

Antagonist-mediated down-regulation of toll-like receptors increases the prevalence of human papillomavirus infection in systemic lupus erythematosus

IntroductionPrevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.MethodsProtein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-γ) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.ResultsFor subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells.ConclusionsIn conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.     

人乳头瘤病毒分型检测在宫颈病变中的研究进展

宫颈癌是感染性疾病,HPV感染是宫颈癌发生的主要因素。宫颈上皮内留样病变(CIN)是宫颈浸润癌演变发展过程中的癌前病变阶段,研究发现大多数CIN伴有人乳头瘤病毒(HPV)感染,不同HPV亚型的致病力不同,因此不同亚型的HPV感染可以导致不同的宫颈病变。对CIN早诊、早治是降低宫颈癌发生率和死亡率的关键,临床上应用HPV亚型检测对宫颈病变的初筛及治疗追踪具有重要意义。     

宫颈癌中端粒酶的表达和高危型人乳头瘤病毒感染

目的：研究不同程度子宫颈病变中高危型人乳头瘤病毒HR-HPV感染和端粒酶活性的表达,以探讨两者在宫颈癌及宫颈上皮内瘤变中的作用及相关性。方法：采用第二代杂交捕获技术检测宫颈脱落细胞HPV．DNA含量,并用免疫组织化学EnVision二步法检测宫颈组织标本中端粒酶的表达。结果：（1）端粒酶阳性表达率在对照组、C1NI、CINII、CINⅢ和宫颈癌组分别为10．00％、16．67％、40．00％、70．00％、95．00％,宫颈癌组高于cINⅢ,CINⅢ高于C1NⅡ,C1NII高于CINI,差异均有统计学意5C（X^2=-4．329,P=0．037;xⅫ．327,P=0．038;X^2=4．022,P=0．045）。（2）随着宫颈病变级别的增加,高危型HPV的阳性率和病毒负荷量均增高。高危型HPV的阳性率在宫颈癌和CINⅢ组明显高于对照组、CINI及CINⅡ（X^2=29．501-7．414,P〈0．01）。高危型HPV的病毒负荷量在对照组与其他4组比较,差异均有统计学意K（P〈0．05）;C1NI组分别与CINⅡ、C1NⅢ及宫颈癌组比较差异均有统计学意义（P〈0．05）。（3）随着宫颈病变级别的增加,高危型HPV的阳性率和端粒酶阳性表达率依次递增,两者有明显的相关性（r=0．943,P〈0．01）。结论：高危型HPV感染和端粒酶活性均与宫颈癌前病变及宫颈癌的发生发展密切相关,有望作为子宫颈癌前病变和宫颈癌筛查的监测指标。