Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.     

土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究

为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR Green I荧光定量PCR检测方法。以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价。结果表明,该方法具有快速、特异性强、敏感度高等特点。检测范围在3.8×103-3.8×108copies/μL之间有良好的线性关系,相关系数R2为0.996,扩增效率为101.5%,灵敏度比常规PCR方法高102倍。     

Protective efficacy of IgM monoclonal antibodies in experimental group B streptococcal infection is a function of antibody avidity

We have produced and characterized six mAb directed against group B streptococci (GBS). All antibodies are IgM. We have previously shown that some of these antibodies are highly protective in the treatment of experimental infections in neonatal rats, whereas others do not appear to have any protective efficacy. Using an ELISA, we demonstrate the specificity of both protective and nonprotective antibodies. Two antibodies, binding different epitopes, are directed against antigenic structures present on all GBS; two are specific for type III carbohydrate determinants; one binds to a protein Ag present on all type I and II GBS; and one appears to bind to type Ia GBS only. Quantitative absorption assays provide evidence that the difference between protective antibodies and nonprotective antibodies is the avidity that the antibody demonstrates for the epitope recognized on the surface of the bacteria; 10 to 15 times as much protective antibody binds to GBS as does nonprotective antibody. Direct binding experiments with radiolabeled antibody confirm this conclusion.     

Chloroplast group III twintron excision utilizing multiple 5''- and 3''-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.     

Histochemical changes in neonatal liver caused by vaginal instillation of magnetic nanoparticles in pregnant mice

Drug delivery through the vagina is a novel and effective approach for treating embryonic diseases. Magnetic nanoparticles (MNPs) currently are used as drug delivery systems. The safety of MNPs for use with embryonic tissues remains unclear. We used pregnant mice to investigate the possible toxicity of MNPs toward neonatal liver at three embryonic ages using histochemical and immunohistochemical techniques. MNPs were instilled through the vaginas of pregnant mice at days 12 (E12), 15 (E15) and 17 (E17) after fertilization. We found MNPs in the neonatal liver parenchyma after delivery of the pups on day 20. We observed that MNPs caused mild apoptosis of hepatocytes, cytoplasmic vacuolation and lymphocytic infiltration in the neonatal liver after treatment at E15 compared to instillation at E12 and E17. We observed also that MNPs increased the production of caspase proteins and tumor necrosis factor receptor 2 proteins, which are indicators of apoptosis, in the neonatal liver after instillation of MNPs at E15 compared to instillation at E12 and E17. MNPs also increased the number of collagen fibers and the amounts of connective tissue growth factors in the neonatal liver parenchyma after instillation at E15 compared to instillation at E12 and E17. The general carbohydrates in the neonatal liver were decreased in a time-dependent manner after instillation at E17, E15 and E12 owing to the presence of MNPs in the parenchyma. Overall, we determined that MNPs were mildly toxic to neonatal liver.