RT-PCR heteroduplex analysis permits differentiation of transgene and host gene expression in a transgenic animal model

In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene. The potential for gene silencing may complicate matters further. Here we report the use of RT-PCR heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences. We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from both human and mouse mRNA samples. Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls were tested. Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples. In addition, RT-PCR heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling. In summary, the RT-PCR heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time. The RT-PCR heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design species-specific RT-PCR primers.     

Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue

Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a approximately 50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated beta-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.     

Fatal outcome of pandemic H1N1 2009 influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice

Pandemic A (H1N1) 2009 influenza virus (pH1N1) infection in pregnant women can be severe. The mechanisms that affect infection outcome in this population are not well understood. To address this, pregnant and nonpregnant BALB/c mice were inoculated with the wild-type pH1N1 strain A/California/04/09. To determine whether innate immune responses are associated with severe infection, we measured the innate cells trafficking into the lungs of pregnant versus nonpregnant animals. Increased infiltration of pulmonary neutrophils and macrophages strongly correlated with an elevated mortality in pregnant mice. In agreement with this, the product of nitric oxide (nitrite) and several cytokines associated with recruitment and/or function of these cells were increased in the lungs of pregnant animals. Surprisingly, increased mortality in pregnant mice was not associated with higher virus load because equivalent virus titers and immunohistochemical staining were observed in the nasal cavities or lungs of all mice. To determine whether exacerbated inflammatory responses and elevated cellularity resulted in lung injury, epithelial regeneration was measured. The lungs of pregnant mice exhibited reduced epithelial regeneration, suggesting impaired lung repair. Despite these immunologic alterations, pregnant animals demonstrated equivalent percentages of pulmonary influenza virus-specific CD8(+) T lymphocytes, although they displayed elevated levels of T-regulator lymphocytes (Tregs) in the lung. Also, pregnant mice mounted equal antibody titers in response to virus or immunization with a monovalent inactivated pH1N1 A/California/07/09 vaccine. Therefore, immunopathology likely caused by elevated cellular recruitment is an implicated mechanism of severe pH1N1 infection in pregnant mice.     

大鼠肺内NOS之分布及缺氧对其活性的影响

本文以组织化学方法对大鼠肺内一氧化氮合酶（ＮＯＳ）进行定位研究,并观察了不同时间（８小时～２８天）缺氧时肺内ＮＯＳ活性的变化。结果显示：①正常大鼠各级支气管、肺泡管和肺泡囊上皮细胞呈ＮＯＳ强阳性反应;肺血管内膜呈ＮＯＳ阳性反应。②缺氧８小时,肺血管内膜ＮＯＳ阳性反应开始降低,并缺氧时间越长,ＮＯＳ阳性反应越低、③缺氧１４天时,肺泡间质和肺血管周围炎性细胞呈ＮＯＳ阳性反应;缺氧２８天时,炎性细胞ＮＯＳ阳性反应增强。④缺氧对支气管、肺泡管和肺泡囊上皮细胞ＮＯＳ的活性无明显影响。从而提示一氧化氮不仅对肺具有一定的生理学作用,而且可能参与缺氧时肺的某些病理学过程。     

Cytotoxic,clonal fidelity,and antioxidant evaluation of in vitro propagated Vitex negundo var. cannabifolia

Plant Cell, Tissue and Organ Culture (PCTOC) - Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. is an important medicinal plant rich in bioactive compounds. In vitro tissue culture...     

Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Regulation of progesterone during follicular development by FSH and LH in sheep

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.