利用假型反转录病毒对大部分胰腺切除后再生细胞的世系追踪

胰腺是一个重要的内外分泌混合腺, 胰腺发生损伤后能够再生。为了探讨胰腺活体细胞世系追踪的方法和胰腺损伤后再生细胞的来源,分别通过胰腺伤口涂抹并胰内注射、尾静脉注射及腹腔注射三种方法, 利用假型反转录病毒对成体小鼠大部分切除后胰腺的细胞进行世系追踪。结果发现在活体条件下, 与尾静脉注射及腹腔注射法相比, 胰腺伤口涂抹并胰腺内注射反转录病毒的方法能够更有效的标记胰腺细胞; 而且, 通过对标记细胞的世系追踪研究证明, 在胰腺损伤后, 胰腺腺泡细胞能够接受损伤信号刺激发生再生。为今后进一步利用反转录假病毒对活体胰腺进行细胞命运追踪研究奠定基础, 为利用反转录病毒载体进行胰腺疾病的基因治疗提供线索。     

Comparison of molecular linkage maps and QTLs for morphological traits in two reciprocal backcross populations of rice

Comparison of maps and QTLs between populations may provide us with a better understanding of molecular maps and the inheritance of traits. We developed and used two reciprocal BC1F1 populations, IP/DS//IP and IP/DS//DS, for QTL analysis. DS (Dasanbyeo) is a Korean tongil-type cultivar (derived from an indica x japonica cross and similar to indica in its genetic make-up) and IP (Ilpumbyeo) is a Korean japonica cultivar. We constructed two molecular linkage maps corresponding to each backcross population using 196 markers for each map. The length of each chromosome was longer in the IP/DS//IP population than in the IP/DS//DS population, indicating that more recombinants were produced in the IP/DS//IP population. Distorted segregation was observed for 44 and 19 marker loci for the IP/DS//IP and IP/DS//DS populations, respectively; these were mostly skewed in favor of the indica alleles. A total of 36 main effect QTLs (M-QTLs) and 15 digenic epistatic interactions (E-QTLs) were detected for the seven traits investigated. The phenotypic variation explained (PVE) by M-QTLs ranged from 3.4% to 88.2%. Total PVE of the M-QTLs for each trait was significantly higher than that of the E-QTLs. The total number of M-QTLs identified in the IP/DS//IP population was higher than in the IP/DS//DS population. However, the total PVE by the M-QTLs and E-QTLs together for each trait was similar in the two populations, suggesting that the two BC1F1 populations are equally useful for QTL analysis. Maps and QTLs in the two populations were compared. Eleven new QTLs were identified for SN, SF, GL, and GW in this study, and they will be valuable in marker-assisted selection, particularly for improving grain traits in tongil-type varieties.     

Refolding and purification of non-fusion HPT protein expressed in Escherichia coli as inclusion bodies

The gene encoding hygromycin B phosphotransferase (hpt) is a widely used selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the non-fusion hpt expression plasmid was constructed and introduced into Escherichia coli to produce enough quantity of the HPT protein. High level expressed HPT was achieved but most of the expressed protein aggregated as inclusion bodies. The inclusion bodies were washed, separated from the cells, and solubilized by 0.3% Sarkosyl. The protein was renatured by dilution and dialysis, and then purified by anion-exchange chromatography. The activity is 8 U/mg protein and the purity is about 95%. Further studies showed that the microbially produced HPT protein had comparable molecular weight, immuno-reactivities, N-terminal amino acid sequences, and biological activities with those of the HPT produced by transgenic rice harboring hpt gene. All these results demonstrated the validity of utilizing the microbially produced HPT to assess the safety of the HPT protein produced in genetically engineered rice.     

Expression of the receptor tyrosine kinase Tie2 in neoplastic glial cells is associated with integrin beta1-dependent adhesion to the extracellular matrix

The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non-stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31- and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin beta1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell-to-extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas.     

Quantifying the Early Immune Response and Adaptive Immune Response Kinetics in Mice Infected with Influenza A Virus

Seasonal and pandemic influenza A virus (IAV) continues to be a public health threat. However, we lack a detailed and quantitative understanding of the immune response kinetics to IAV infection and which biological parameters most strongly influence infection outcomes. To address these issues, we use modeling approaches combined with experimental data to quantitatively investigate the innate and adaptive immune responses to primary IAV infection. Mathematical models were developed to describe the dynamic interactions between target (epithelial) cells, influenza virus, cytotoxic T lymphocytes (CTLs), and virus-specific IgG and IgM. IAV and immune kinetic parameters were estimated by fitting models to a large data set obtained from primary H3N2 IAV infection of 340 mice. Prior to a detectable virus-specific immune response (before day 5), the estimated half-life of infected epithelial cells is ∼1.2 days, and the half-life of free infectious IAV is ∼4 h. During the adaptive immune response (after day 5), the average half-life of infected epithelial cells is ∼0.5 days, and the average half-life of free infectious virus is ∼1.8 min. During the adaptive phase, model fitting confirms that CD8⁺ CTLs are crucial for limiting infected cells, while virus-specific IgM regulates free IAV levels. This may imply that CD4 T cells and class-switched IgG antibodies are more relevant for generating IAV-specific memory and preventing future infection via a more rapid secondary immune response. Also, simulation studies were performed to understand the relative contributions of biological parameters to IAV clearance. This study provides a basis to better understand and predict influenza virus immunity.Current strategies for preventing or decreasing the severity of influenza infection focus on increasing virus-neutralizing antibody titers through vaccination, as experience indicates that this is the best way to prevent morbidity and mortality. Influenza A virus (IAV) undergoes mutations of the genes encoding the hemagglutinin (HA) and neuraminidase (NA) proteins that the neutralizing antibodies are directed against. When the variation is low (antigenic drift), prior vaccination often confers substantial heterologous immunity against a new seasonal IAV strain. In contrast, major genetic changes (antigenic shift) can result in pandemic IAV strains, since for novel strains, the humoral immune response is a primary response, and heterologous immunity is lacking. The emergence of such pandemic strains and the fact that young children are more vulnerable to influenza diseases highlight the need to better understand which viral and immune parameters determine the outcome of infection with viruses novel to the individual.Conventional experimental methods to measure influenza virus immunity have been limited to animal models and studies of adult human peripheral blood leukocytes. The advantages of using animal models include the ability to intensively sample multiple tissues and to utilize genetic and other interventions, such as blocking or depleting antibodies, to dissect the contribution of individual arms of the immune system. However, it is easy to question the relevance of these experiments to humans because of the many important biological differences between human and murine immune systems (29). In both the animal and human systems, we are limited to measuring those parameters and variables for which assays are available, most of them being ex vivo. Parameters such as cell-to-cell spread of the virus in vivo, trafficking of immune cells to the lung, and the in vivo interactions in an intact immune system are much more difficult or impossible to measure with contemporary techniques, particularly in humans. Computational approaches have the potential to offset some of these limitations and provide additional insight into the kinetics of the IAV infection and the associated immune response.Animal models of influenza virus infection in which different arms of the immune system have been suppressed suggest that some components of the adaptive immune system are required for complete viral clearance, often termed a sterilizing immune response. For example, abrogation of the CD4 T-cell response by cytotoxic antibody therapy or through knockout of major histocompatibility complex (MHC) class II slightly delays viral clearance but has little overall effect on the ability to control the infection (21, 54, 55). Elimination of the CD8 T-cell response typically results in delayed viral clearance (12, 20, 47), although animals with intact CD4 T-cell and B-cell compartments are able to control the infection in the absence of CD8 T cells. Presumably, this occurs through antibody-mediated mechanisms (54). Most animals depleted of both CD8 T cells and B cells are not able to clear the virus, which results in death (14, 32, 53). CD4⁺ T cells certainly contribute to the control of IAV infection, although cytotoxic CD4 T cells are not frequently observed unless cultured in vitro (8, 22, 45). Thus, it is generally accepted that CD8 T cells and/or antibodies are sufficient for timely and complete IAV clearance. Studies that strictly separate the relative roles of CD8 T cells and virus-specific antibodies are less satisfying. Animals depleted of both CD4 and CD8 T cells generally do not control the infection, despite substantial production of anti-IAV IgM antibodies (4, 23, 33, 34). However, adoptive transfer of IAV-specific IgM or IgG antibodies is protective (40, 51), suggesting that the timing and magnitude of the antibody response, i.e., the affinity, avidity, and antibody isotype, are important protective factors.While murine gene knockout or lymphocyte depletion studies are highly informative, they also have a number of limitations. Most importantly, the near-complete ablation of one component of the adaptive immune system often causes profound and unpredictable effects on many other immune components. Although the reported experimental measurements are highly quantitative, they often focus only on a limited number of time points or measurements and do not capture the complexity of the altered, or intact, immune response. In contrast, high-frequency experimental sampling, coupled with mathematical modeling techniques and new statistical approaches, can give insights into the complex biology of IAV infection and test the assumptions inherent in the model. We have learned from other systems, particularly HIV (19, 35, 37, 38, 56), that quantitative analysis of the biology can reveal important factors that are not intuitively obvious. For example, our current estimates for the rates of HIV production and the life span of productively infected cells in vivo were obtained via mathematical modeling (35).Mathematical models have long been used to investigate viral dynamics and immune responses to viral infections, including influenza A virus (3, 5, 7, 15, 16, 31, 36, 48). We recently described a complex differential equation model to simulate and predict the adaptive immune response to IAV infection (24). This model involves 15 equations and 48 parameters, and because of its complexity, many of the parameter values that could not be directly measured were unidentifiable. Thus, it is difficult to estimate all model parameters by fitting experimental data directly to this complex model, although the model can be used to perform simulation predictions (25). This issue can, however, be addressed by reducing the model into smaller submodels with smaller but identifiable sets of parameters, which can be estimated from experimental data. In this paper, we describe such an approach which focuses on IAV infection and the immune response solely within the lung.In the present report, we have fitted a model of primary murine influenza virus infection to data. In naïve subjects, our data suggested that there is no adaptive immune response (e.g., IAV-specific CD8⁺ T cells or antibodies) detectable in the spleen, lymph nodes, or lung until approximately 5 days after infection; therefore, we have divided the analysis into the following two phases: the initial preadaptive (innate) phase and the later adaptive phase. We use direct experimental data from infection of mice with the H3N2 influenza virus A/X31 strain (2, 24) to obtain key kinetic parameters. The model fitting has revealed that the duration of the infection depends on a small set of immune components, and even large fluctuations in other arms of the immune system or IAV behavior have surprisingly little impact on the outcome of the infection.     

Effects of combined administration of quercetin, rutin, and extract of white radish sprout rich in kaempferol glycosides on the metabolism in rats

Quercetin, rutin, the extract of white radish sprout rich in kaempferol glycosides, and their combination were intragastrically administered to Wistar rats to investigate the interactive metabolism of these flavonoids. The combined administration of these flavonoids changed the concentrations of the metabolites in plasma as compared with the concentrations after the administration of a single compound.     

LncRNA KCNQ1OT1 activated by c-Myc promotes cell proliferation via interacting with FUS to stabilize MAP3K1 in acute promyelocytic leukemia

Uncontrolled proliferation is the hallmark of cancer cells. Previous studies mainly focused on the role of protein-coding genes in cancer cell proliferation. Emerging evidence showed that long non-coding RNAs (lncRNAs) also play critical roles in cancer cell proliferation and growth. LncRNA KCNQ1OT1 is found to contribute to carcinogenesis, but its role in acute promyelocytic leukemia (APL) is unclear. In this study, by analyzing data from Gene Expression Omnibus, The Cancer Genome Atlas database and our clinical samples, we found that KCNQ1OT1 was selectively highly expressed in APL. Functional assays demonstrated that knockdown of KCNQ1OT1 reduced APL cell proliferation and increased apoptosis. Further evidence showed that KCNQ1OT1 was mainly located in the cytoplasm of APL patient-derived NB4 cells and APL patient bone marrow samples. Mechanistically, KCNQ1OT1 bound to RNA binding protein FUS, and silencing either KCNQ1OT1 or FUS reduced the expression level and stability of MAP3K1 mRNA. Whereas KCNQ1OT1 and FUS did not affect each other. Importantly, knockdown of MAP3K1 impaired APL cell proliferation. Finally, c-Myc transactivated KCNQ1OT1 in APL cells through binding to its promoter while knockdown of c-Myc decreased KCNQ1OT1 expression. Our results not only revealed that c-Myc transactivated KCNQ1OT1 and upregulated KCNQ1OT1 promoted APL cell proliferation, but also demonstrated that KCNQ1OT1 bound to FUS to synergistically stabilize MAP3K1 mRNA, thus facilitating APL cell proliferation. This study established a previously unidentified role of KCNQ1OT1 in the development of APL, and KCNQ1OT1 may serve as a potential therapeutic target for APL.Subject terms: Acute myeloid leukaemia, Acute myeloid leukaemia     

Aspergillus niger DLFCC-90 rhamnoside hydrolase, a new type of flavonoid glycoside hydrolase

A novel rutin-α-L-rhamnosidase hydrolyzing α-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13.     

Protective effect of ginseng sapogenins against 2,2'-azobis (1-aminopropane) dihydrochloride (AAPH)-induced LLC-PK₁ cell damage

The effect of ginseng sapogenins, aglycone parts of ginsenosides, against oxidative damage by radical generator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), in renal epithelial LLC-PK(1) cells was investigated to identify the structural characteristics of sapogenins to have renoprotective effects. Of the tested sapogenins, Δ(20(21))-protopanaxatriol showed the strongest protective effect against the AAPH-induced LLC-PK(1) cell damage. Based on the structure and stronger activity of Δ(20(21))-protopanaxatriol than the other sapogenins, the hydroxyl group in C-6 and double bond in C-20(21) position were important for renoprotective effect of sapogenin against oxidative stress.