Heavy metal-mediated crystallization of Escherichia coli phytase and analysis of bridging interactions

Escherichia coli phytase is a phosphatase that catalyzes the hydrolysis of phytic acid into inorganic phosphate and myo-inositol. Two crystal forms of this enzyme were obtained in the presence of heavy metals. Crystal forms I and II were obtained with the heavy atoms CdCl(2) and HgCl(2) and diffracted to 1.5 A and 2.25 A resolution, respectively. Hg(2+) and Cd(2+) both acted as molecular bridge(s), linking and stabilizing E. coli phytase in the unit cell, and played a crucial role in the crystallization of phytase by bridging neighbouring symmetry related molecules.     

High mortality due to Tetrahymena sp. infection in laboratory-maintained zebrafish (Brachydanio rerio)

A large colony of laboratory zebrafish (Brachydanio rerio) used in the study of early vertebrate embryogenesis began experiencing acute, unexplained mortality that approached 100% among approximately 30-day-old resident fry. The initial differential diagnosis included ammonia, nitrite, or chlorine toxicosis, as well as iatrogenically induced toxicosis associated with improper sanitation procedures of laboratory equipment. Necropsy of dead and moribund fry prior to fixation revealed swarms of ovoid-shaped, motile, ciliated protozoa with a "spiraling football" motion. Wet mount preparations of various water samples also contained high numbers of similar protozoa. Histologic examination of affected fry revealed numerous, periodic acid-Schiff-positive forms within the body coelom, and epithelial and muscle tissues. The protozoa were consistent morphologically with members of the genus Tetrahymena, which is usually a free-living, nonpathogenic ciliated protozoa in fresh and saltwater environments. Relevant disease associated with Tetrahymena spp. in viviparous fish has been reported as a result of concurrent disease, immunosuppression, or poor water quality conditions. To the authors' knowledge, this is the first report of an epizootic involving laboratory maintained zebrafish, and the diagnostic course and therapeutic interventions undertaken to alleviate Tetrahymena species-associated clinical disease.     

Sensitive liquid chromatographic--mass spectrometric assay for norfloxacin in poultry tissue

A highly sensitive and specific method for the determination of norfloxacin in poultry tissues by LC-MS was developed and validated. An extract of the sample was separated on a C(18) reversed-phase column and analyzed by LC-MS. The mobile phase was gradiently flowed with 2% acetic acid and acetonitrile. The limit of detection and limit of quantitation were 1 and 5 ng/g, respectively. Mean recoveries from various spiked tissues were 87.2% (ranging from 82.5 to 92.7%) for norfloxacin. The method has been successfully applied to determine norfloxacin in poultry muscle.     

High-throughput screening of potential inhibitors for the metabolism of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid

By screening potential inhibitors of drug metabolism using the in vitro models, potential drug-drug interactions in vivo may be predicted with the use of appropriate pharmacokinetic principles. This study aimed to develop a rapid screening system using human liver microsomes to efficiently identify the potential inhibitors of DMXAA metabolism. Initial IC50 was estimated by using a two-point method, and then Ki values were determined if required and compared with those initial IC50 values. More than 100 compounds including known substrates and inhibitors of human uridine diphosphate glucuronosyltransferases (UGTs) and cytochrome P450 (CYP), anti-cancer drugs and xanthenone analogues were screened for their inhibitory effect on DMXAA glucuronidation and 6-methylhydroxylation in human liver microsomes. Both metabolites of DMXAA, DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA), formed in human liver microsomes were quantitated by validated HPLC methods. The results indicated that there was a significant relationship (r2 = 0.966, P < 0.001) between the two-point IC50 values and the apparent Ki values for 20 compounds showing significant inhibitory effects on DMXAA metabolism, suggesting the usefulness of the two-point determination for the initial screening of compounds. This study has been completed using a strategy for rapid HPLC analysis and thus provided early access to detailed information for potential inhibitors of DMXAA metabolism and allows for further DMXAA-drug interaction studies.     

Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into a variety of cell types, which could be used for cell transplantation therapies as well as drug discovery studies. However, the large-scale culture of undifferentiated human ES cells is currently limited by their dependency on mouse embryonic fibroblast feeder layers. The proteomics approach was employed to characterize the environment that supports the growth of undifferentiated human ES cells and to identify factors critical for their independent growth. Conditioned medium from mouse embryonic fibroblast feeder layers, STO cell line, was concentrated and subjected to analyses by two-dimensional electrophoresis mass spectrometry. In total, 136 unique protein species were identified which included some that are known to participate in cell growth and differentiation, extracellular matrix formation and remodeling, in addition to the unexpected but interesting finding of many nominally intracellular proteins. This approach has thus revealed the complexity of the environment provided by the feeder cells and provides a useful starting point for future studies. Moreover, candidates from the initial list of identified proteins can be further investigated for their effects on the growth and differentiation of human ES cells in a defined culture environment.     

Whole bone marrow transplantation induces angiogenesis following acute ischemia

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia. BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa 26 (Rosa 26) mice that express beta-galactosidase (beta-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 micro l containing 1 x 10(6) BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 micro l of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived beta-gal(+) cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation. Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.     

Multiple virus resistance in transgenic plants conferred by the human dsRNA-dependent protein kinase

We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM _r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants.