Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis

Ubiquitin-mediated protein modification plays a key role in many cellular signal transduction pathways. The Arabidopsis gene XBAT32 encodes a protein containing an ankyrin repeat domain at the N-terminal half and a RING finger motif. The XBAT32 protein is capable of ubiquitinating itself. Mutation in XBAT32 causes a number of phenotypes including severe defects in lateral root production and in the expression of the cell division marker CYCB1;1::GUS . The XBAT32 gene is expressed abundantly in the vascular system of the primary root, but not in newly formed lateral root primordia. Treatment with auxin increases the expression of XBAT32 in the primary root and partially rescues the lateral root defect in xbat32 - 1 mutant plants. Thus, XBAT32 is a novel ubiquitin ligase required for lateral root initiation.     

Synthesis of chiral phosphoantigens and their activity in gamma delta T cell stimulation

Gammadelta T cells expressing Vgamma2Vdelta2 T cell receptors are activated by a broad range of phosphorus-containing small molecules, termed phosphoantigens, and are of interest in the context of the chemotherapy of B cell malignancies. Here, we report the synthesis of four pairs of chiral phosphoantigens: the bromohydrins of isopentenyl diphosphate (Phosphostim), the epoxides of isopentenyl diphosphate (EIPP); and the corresponding bromohydrin and epoxide analogs of but-3-enyl diphosphate. The ability of each compound to stimulate human Vgamma2Vdelta2 T cells was determined by TNF-alpha release and cell proliferation. In these assays, the (R)-bromohydrin diphosphates were, on average, about twice as active as the (S)-bromohydrin diphosphates. In contrast, the (S)-form of EIPP was about twice as active as (R)-EIPP. The activities of the epoxy but-3-enyl diphosphates were both very low. These results suggest that chiral phosphoantigens, as opposed to racemic mixtures, may have utility in immunotherapy.     

Peripheral substituents of di(pyridiumyl)porphyrins affected on their interactions with DNA

meso- and beta-Substituted di(pyridiumyl)porphyrins 3, 4, and 7 have been synthesized and their interactions with DNA have been investigated. meso-Substituted porphyrins showed the stronger effect on DNA than that of beta-substituted porphyrin. Cytotoxicity of compound 3 (IC(50)) to THP-1 tumor cell was up to 0.11 nM.     

The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants

All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

A generalized likelihood ratio test to identify differentially expressed genes from microarray data

MOTIVATION: Microarray technology emerges as a powerful tool in life science. One major application of microarray technology is to identify differentially expressed genes under various conditions. Currently, the statistical methods to analyze microarray data are generally unsatisfactory, mainly due to the lack of understanding of the distribution and error structure of microarray data. RESULTS: We develop a generalized likelihood ratio (GLR) test based on the two-component model proposed by Rocke and Durbin to identify differentially expressed genes from microarray data. Simulation studies show that the GLR test is more powerful than commonly used methods, like the fold-change method and the two-sample t-test. When applied to microarray data, the GLR test identifies more differentially expressed genes than the t-test, has a lower false discovery rate and shows more consistency over independently repeated experiments. AVAILABILITY: The approach is implemented in software called GLR, which is freely available for downloading at http://www.cc.utah.edu/~jw27c60     

Experimental study of rat beta islet cells cultured under simulated microgravity conditions

To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of the cells was observed by electron microscopy. Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured under the microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient trans     

Expression of a modified Cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer

The wild-type Crylle gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Crylle gene (designated as Cryllem) was cloned into prokaryotic expressionvector pET28b and its expression in E.coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E.coli revealed that CrylIem protein had a similar toxicity to corn borer as wild-type CrylIe. CrylIem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cryllem showed insecticidal activity against corn borer.