Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.     

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn²⁺) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn²⁺ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl₂ markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn²⁺ has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-l-cysteine (NAC) blunted the increase in Zn²⁺ levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn²⁺ contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.     

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.     

Allosteric inhibition of zinc-finger binding in the major groove of DNA by minor-groove binding ligands

In recent years, two methods have been developed that may eventually allow the targeted regulation of a broad repertoire of genes. The engineered protein strategy involves selecting Cys(2)His(2) zinc finger proteins that will recognize specific sites in the major groove of DNA. The small molecule approach utilizes pairing rules for pyrrole-imidazole polyamides that target specific sites in the minor groove. To understand how these two methods might complement each other, we have begun exploring how polyamides and zinc fingers interact when they bind the same site on opposite grooves of DNA. Although structural comparisons show no obvious source of van der Waals collisions, we have found a significant "negative cooperativity" when the two classes of compounds are directed to the overlapping sites. Examining available crystal structures suggests that this may reflect differences in the precise DNA conformation, especially with regard to width and depth of the grooves, that is preferred for binding. These results may give new insights into the structural requirements for zinc finger and polyamide binding and may eventually lead to the development of even more powerful and flexible schemes for regulating gene expression.     

Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera

AIMS: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. METHODS AND RESULTS: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. CONCLUSIONS: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.     

Chirality effect of C-20 on metabolism of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives

Epimers at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oates, their 9alpha-fluoro analogs, their carbonate derivatives, and their acetonide derivatives were subjected to metabolism study in rat plasma and rat liver homogenate. These steroids were synthesized based on the antedrug concept. In rat plasma, the carboxy ester bonds of 20beta-triols and their acetonides were hydrolyzed with half-lives (T(1/2)) of between 5.7 and 7.7 min, while their corresponding alpha-epimers had longer half-lives of more than 2.5 h. A more profound difference was observed between the alpha- and beta-epimers of the carbonates, with the latter showing a T(1/2) less than 1 min (0.3 and 0.43 min for P20beta- and PF20beta-carbonate, respectively), while that of the former about 3 h (165 min for P20alpha-carbonate and 191 min for PF20alpha-carbonate). In rat liver homogenate, the triol and acetonide derivatives showed greater stability than they did in rat plasma, with T(1/2) for the beta-group in the range of 54-108 min, and T(1/2) for the alpha-group over 7 h. A significant difference in hydrolysis of the carbonate derivatives was also observed in rat liver homogenate. The half-lives of P20beta- and PF20beta-carbonate were 0.67 and 0.66 min, respectively, and the alpha-isomers showed the similar metabolic rate with other alpha-isomers. An esterase inhibitor effectively blocked the hydrolysis of the ester bond, indicating that this metabolism is an enzymatic reaction. Molecular modeling studies show that steric hindrance around the ester group of the alpha-epimers is much greater than that of their beta-counterparts, affording one explanation for the large difference in the metabolic hydrolysis rate; i.e. the carboxy ester bond of beta-isomer which is less hindered sterically than their counter alpha-isomers is hydrolyzed faster than that of alpha-isomers. In conclusion, this study confirms that chirality at C-20 had profound effects on metabolism and pharmacological profile of the steroid acid ester derivatives.