Dinucleotide polymorphism of p73 gene is associated with a reduced risk of lung cancer in a Chinese population

p73, a structural and functional homologue of p53, shares some p53-like tumor suppressor activity but also possesses oncogenic activity. Therefore, p73 plays an important role in modulating cell-cycle control and apoptosis. A potentially functional dinucleotide polymorphism, G4C14-to-A4T14, has been identified in the 5' untranslated region (UTR) of exon 2 of the p73 gene, which may theoretically form a stem-loop structure and affect gene expression. To test the hypothesis that these 2 common variants play a role in lung cancer susceptibility, we conducted a case-control study of 425 lung cancer patients and 588 cancer-free controls frequency-matched to the cases on age and sex in a Chinese population. The results showed that these 2 polymorphisms were in complete linkage disequilibrium and the frequencies of variant p73 AT haplotype (A4T14) were less common in the cases (0.225) than in the controls (0.287) (p = 0.0018), suggesting that this AT haplotype was protective against lung cancer. Compared to the p73 GC/GC homozygotes, both the AT/AT variant homozygotes and GC/AT heterozygotes were associated with a significantly decreased risk (adjusted OR: 0.45, 95% CI: 0.26-0.80 and OR: 0.70, 95% CI: 0.53-0.92, respectively). These results suggest that this p73 dinucleotide polymorphism may have a role in lung cancer susceptibility in our study population. Further studies are needed to elucidate potential functional relevance of the p73 AT variant allele.     

CD 39-associated high ATPase activity contribute to the loss of P 2 X 7-mediated calcium response in LCL cells

The P 2 X 7 nucleotide receptor is an adenosine 5'-triphosphate (ATP)-gated ion channel, which induces cation channel opening imparting significant permeability to Ca(2+), and is widely expressed in cells of hematopoietic origin. Our previous report showed that P 2 X 7-mediated calcium response was absent in three Epstein-Barr virus (EBV)-positive and P 2 X 7 positive cell lines. In this report, we detected the cell surface ATPase activity, which contributes to the hydrolysis of extracellular ATP, and the expression of CD 39, which is the main source of ATPase on hematopoietic cells, in these cell lines. Then, we tried to restore the P 2 X 7-mediated calcium response in LCL-H and J 6-1 cells by either increasing the concentration of agonist or suppressing the ATPase activity by betagammaMeATP, a synthetic poorly metabolizable ATP analogue. The results showed that LCL-H and J 6-1 cells had higher levels of ATPase activity and CD 39 expression. The treatment of 300 microM betagammaMeATP efficiently inhibited the ATPase activity on LCL-H and J 6-1 cells. Both elevation of agonist concentration (10mM ATP or 1mM BzATP) and pretreatment with 300 microM betagammaMeATP followed by stimulation with normal concentration of agonists (1mM ATP or 0.1mM BzATP) could cause P 2 X 7-mediated calcium response in LCL-H but neither in J 6-1 cells. These results suggested that multiple mechanisms contributed to the loss of the P 2 X 7-mediated calcium response. CD 39-associated high ATPase activity contributed to the loss of the P 2 X 7-mediated calcium response in LCL-H cells, while additional mechanism(s) existed in J 6-1 cells.     

胃癌腹膜高转移细胞特异性结合噬菌体多肽的筛选及鉴定

目的寻找能够与胃癌腹膜高转移细胞GC9811-P特异性结合的噬菌体多肽,探索治疗胃癌腹膜转移的新方法。方法运用噬菌体呈现肽技术,先后用胃癌的腹膜高转移细胞系GC9811-P和其亲本细胞GC9811对噬菌体12肽库进行消减性的全细胞淘洗．经过3轮筛选,随机挑选40个噬菌体单克隆C1～G40。用ELISA法选取能够与GC9811-P特异性结合的单克隆。将选出的单克隆分别注入裸鼠腹腔,采用免疫组化法排除与正常组织亦高结合的阳性单克隆。对筛选出的噬菌体克隆进行DNA序列测定,并推导其外源性氨基酸序列,进行同源性分析。结果经过3轮淘洗,噬菌体克隆得到理想富集。C9、C18、C23、C29、C34和C37可与GC9811-P特异性结合,经免疫组化证实,这6个单克隆均不与裸鼠腹腔内正常组织结合。测序结果大致展示了两种外源性多肽,即TLNINRLIIPRT和SMSIxSPYIxxx。结论筛选出6个可与GC9811-P细胞特异性结合的噬菌体多肽;这两个肽序列能否阻断GC9811-P细胞向腹膜转移尚待进一步确定。     

Structure-based design, synthesis, and study of potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, beta-ketoacyl-ACP synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Small molecules that inhibit FabH enzymatic activity have the potential to be candidates within a novel class of selective, nontoxic, broad-spectrum antibacterials. Using crystallographic structural information on these highly conserved active sites and structure based drug design principles, a benzoylaminobenzoic acid series of compounds was developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity against Gram-positive and selected Gram-negative organisms.     

Protection of esophageal multi-lineage progenitors of squamous epithelium (stem cells) from ionizing irradiation by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy

BACKGROUND: Intraesophageal manganese superoxide dismutase plasmid liposome (MnSOD-PL) gene therapy protects against irradiation damage. MATERIALS AND METHODS: To determine whether esophageal side population (SP) stem cells were protected, epitope-tagged (hemagglutinin) (HA) MnSOD-PL was administered to C57BL/6J mice 24 hours prior to 30 Gy esophageal irradiation. SP cells were isolated, and apoptosis and multi-lineage vimentin/endothelin/F4/80 (macrophage) colonies in vitro were quantitated. RESULTS: The number and percent of SP cells, apoptotic cells, or numbers of multi-lineage vimentin/endothelin/F4/80-positive in vitro colonies isolated from non-irradiated HA-MnSOD-PL-treated or 30 Gy-irradiated esophagus did not differ between groups. Irradiation in vitro significantly increased apoptosis in explanted non-SP cells from control (p = 0.021) compared to MnSOD-PL-treated mice. Irradiation-induced cell division was significantly increased in SP cells from control-irradiated mice (p = 0.001), but not MnSOD-PL-treated mice. Irradiation-induced apoptosis detected in vivo at 5 days was decreased by MnSOD-PL. CONCLUSION: MnSOD-PL gene therapy protects esophageal SP cells from irradiation in vitro and in vivo.     

DC-SIGN： Binding receptor for HCV？

DC-SIGN, a dendritic Cell-specific adhesion receptor and a type Ⅱ transmembrane mannose-binding C-type lectin, is very important in the function of DC, both in mediating naive T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by viral and bacterial pathogens including Human Immunodeficiency Virus (HIV), HCV, Ebola Virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in bans, by transmission of bound virus to a target cell expressing appropropriate entry receptors. Recent work showed that DC-SIGN are highaffinity binding receptors for HCV. Besides playing a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental for the interaction of DC with T cells during antigen presentation. The clinical strategies that target DCSIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.     

Prevalence of subclinical hepatic encephalopathy in cirrhotic patients in China

AIM: Subclinical hepatic encephalopathy (SHE) is a common complication of liver diseases. The aim of this study was to find out the normal value of psychometric test and to investigate the prevalence of SHE in Chinese patients with stabilized hepatic cirrhosis.METHODS: Four hundred and nine consecutive cirrhotic patients without overt clinical encephalopathy were screened for SHE by using number connection test part A (NCT-A) and symbol digit test (SDT). SHE was defined as presence of at least one abnormal psychometric test. The age-corrected normal values were defined as the mean±2times standard deviation (2SD), and developed in 356 healthypersons as normal controls. Four hundred and sixteen patients with chronic viral hepatitis were tested as negative controls to assess the diagnostic validity of this test battery.RESULTS: There was no significant difference in NCT scores and SDT quotients between healthy controls and chronic hepatitis group (P>0.05). In all age subgroups,the NCT and SDT measurements of cirrhotic patients differed significantly from those of the controls (P<0.05).When mean±2SD of SDT and NCT measurements from healthy control group was set as the normal range, 119 cirrhotic patients (29.1%) were found to have abnormal NCT-A and SDT tests, 53 (13.0%) were abnormal only in SDT and 36 (8.8%) only in NCT-A. Taken together, SHE was diagnosed in 208 (50.9%) cirrhotic patients by this test battery. The prevalence of SHE increased from 39.9% and 55.2% in Child-Pugh‘s grade A and B groups to 71.8% in Child-Pugh‘s grade C group (P<0.05). After the adjustment of age and residential areas required from the tests, no correlation was found in the rate of SHE and causes of cirrhosis, education level and smoking habit.CONCLUSION: Psychometric tests are simple and reliable indicators for screening SHE among Chinese cirrhotic patients. By using a NCT and SDT battery, SHE could be found in 50.9% of cirrhotic patients without overt clinical encephalopathy. The prevalence of SHE is significantly correlated with the severity of liver functions.     

抗人Apo B100单克隆抗体的制备及夹心ELISA检测方法的建立

目的:制备抗人载脂蛋白B100(Apo B100)单克隆抗体(mAb),建立人Apo B100双抗体夹心ELISA检测方法。方法:将人Apo B100抗原免疫小鼠,通过细胞融合、筛选后得到杂交瘤细胞株。将细胞株用无血清培养基扩大培养并纯化上清获得抗体,测定抗体亲和力、亚型、特异性及表位,最后建立双抗体夹心ELISA方法。结果:获得4株抗人Apo B100的杂交瘤细胞株(4-1-2、4-2-2、4-3-2、4-6-3),其分泌的抗体不与其他相关蛋白交叉反应,亲和力达到1×109L/mol。用4-3-2和4-6-3建立的双抗体夹心法的检测范围为(1.3～80)ng/mL,灵敏度1.24 ng/mL,批内变异系数均小于10%,批间变异系数均小于15%,回收率在90%以上。结论:成功制备了抗人Apo B100mAb,建立了定量检测人Apo B100的双抗体夹心ELISA方法,为Apo B100检测及疾病的诊断奠定基础。     

Lipopolysaccharide mediated mast cells induce IL-10 producing regulatory T cells through the ICOSL/ICOS axis

The mechanisms by which mast cells (MCs) regulate immune responses are still largely unknown. Here, we showed that MCs induced interleukin (IL)-10 producing T cells to regulate inflammatory responses. To gain insight into the molecules involved, we set up an in vitro system in which lipopolysaccharide (LPS) stimulated MCs and CD4(+) T cells were co-cultured. Induction of IL-10 producing regulatory T cells mainly relied on the inducible costimulator ligand (ICOSL)/ICOS axis. MCs stimulated with LPS for more than 6 weeks upregulated ICOSL expression, while icosl(-/-) BMMCs failed to induce IL-10 producing T cells. The LPS effect was mediated through NF-κB activation via the TLR4 signaling pathway. Ex vivo analysis of bronchoalveolar lavage fluid from mice with LPS-mediated pneumonia revealed ICOSL(+) MCs and IL-10 producing T cell induction. Additionally, adaptive transfer of ICOSL(+) BMMCs attenuated LPS-mediated inflammation in MC-deficient mice. This study provided new evidence for the regulatory role of MCs.     

MicroRNA-193 Pro-Proliferation Effects for Bone Mesenchymal Stem Cells After Low-Level Laser Irradiation Treatment Through Inhibitor of Growth Family, Member 5

The enhanced proliferation of mesenchymal stem cells (MSCs) can be helpful for the clinical translation of cell therapy. Low-level laser irradiation (LLLI) has been demonstrated as regulating MSC proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in stem cells, but the role of miRNAs in the LLLI-based promotion of MSC proliferation remains unclear. We found that the proliferation level and cell cycle-associated genes in MSCs were increased after LLLI treatment in a time-dependent manner. Microarray assays revealed subsets of miRNAs to be differentially regulated, and these dynamic changes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) after LLLI. miR-193 was the most highly up-regulated miRNA, and the change in it was related with the proliferation level. Gain-loss function experiments demonstrated that miR-193 could regulate the proliferation of MSCs, including human's and rat's, but could not affect the apoptosis and differentiation level. Blockade of miR-193 repressed the MSC proliferation induced by LLLI. By qRT-PCR, we found that miR-193, in particular, regulated cyclin-dependent kinase 2 (CDK2) expression. Bioinformatic analyses and luciferase reporter assays revealed that inhibitor of growth family, member 5 (ING5) could be the best target of miR-193 to functionally regulate proliferation and CDK2 activity, and the mRNA and protein level of ING5 was regulated by miR-193. Furthermore, the ING5 inhibited by small interfering RNA (siRNA) could up-regulate the proliferation of MSCs and the expression of CDK2. Taken together, these results strongly suggest that miR-193 plays a critical part in MSC proliferation in response to LLLI stimulation, which is potentially amenable to therapeutic manipulation for clinical application.