Plasmid vectors harboring cellular promoters can induce prolonged gene expression in hematopoietic and mesenchymal progenitor cells

Although prolonged transgene expression in progenitor cells might be desirable for modified cell therapy, the viral promoter-based expression vector tends to promote transgene expression only for a limited period. Here, we examined the ability of cellular promoters from elongation factor-1alpha (EF-1alpha) and ubiquitin C to drive gene expression in hematopoietic TF-1 and mesenchymal progenitor cells. We compared the expression levels and duration of a model gene, interleukin-2, generated by the cellular promoters to those by the cytomegalovirus (CMV) promoter. The EF-1alpha and ubiquitin C promoters drove prolonged gene expression in hematopoietic TF-1 and mesenchymal progenitor cells, whereas the CMV promoter did not. At day 7 after transfection in TF-1 cells, the mRNA expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 118- and 56-fold higher, respectively, than those driven by the CMV promoter. Similarly, in mesenchymal progenitor cells, the expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 98- and 20-fold higher, respectively, than that driven by the CMV promoter-encoding plasmid. Moreover, the ubiquitin C promoter directed higher levels of green fluorescence protein expression in mesenchymal progenitor cells than did the CMV promoter. These results indicate that the use of cellular promoters such as those for EF-1alpha and ubiquitin C might direct prolonged gene expression in hematopoietic and mesenchymal progenitor cells.     

Effect of N-Acetylcystein on butyrate-treated Chinese hamster ovary cells to improve the production of recombinant human interferon-beta-1a

Sodium butyrate (NaBu) is used as a productivity enhancer for the production of therapeutic recombinant proteins in Chinese hamster ovary (CHO) cells. However, NaBu is well-known for having a cytotoxic effect, thereby inducing apoptosis. As an endeavor to reduce this defect, we studied 11 antioxidants known for inhibiting apoptosis, according to a Plackett-Burman statistical design on CHO cells producing recombinant interferon-beta-1a (IFN-beta). None of the antioxidants that we tested were as effective as N-acetylcystein (NAC) from the point of view of maintaining long-term survival of CHO cells and increasing the production of IFN-beta. In 7.5-L perfusion bioreactor cultures, the addition of NaBu and NAC elongated the culture period to almost 200 h throughout production phase and increased the production yield by 2-fold compared to control cultures containing only NaBu. Glycosylation patterns of produced IFN-beta at each run were also compared in IEF analysis. IEF profiles of where NaBu and NAC were added showed to be more isoforms with a lower pI than those of the control run. The sialic acid content was also increased by 17.7% according to HPLC analysis. Taken together, the data obtained demonstrate that the addition of NAC has positive effects on the elongation of the culture period, improving the production and increasing the sialylation of IFN-beta in NaBu-treated CHO cells.     

Predicting genes expressed via -1 and +1 frameshifts

Computational identification of ribosomal frameshift sites in genomic sequences is difficult due to their diverse nature, yet it provides useful information for understanding the underlying mechanisms and discovering new genes. We have developed an algorithm that searches entire genomic or mRNA sequences for frameshifting sites, and implements the algorithm as a web-based program called FSFinder (Frameshift Signal Finder). The current version of FSFinder is capable of finding -1 frameshift sites on heptamer sequences X XXY YYZ, and +1 frameshift sites for two genes: protein chain release factor B (prfB) and ornithine decarboxylase antizyme (oaz). We tested FSFinder on approximately 190 genomic and partial DNA sequences from a number of organisms and found that it predicted frameshift sites efficiently and with greater sensitivity and specificity than existing approaches. It has improved sensitivity because it considers many known components of a frameshifting cassette and searches these components on both + and - strands, and its specificity is increased because it focuses on overlapping regions of open reading frames and prioritizes candidate frameshift sites. FSFinder is useful for discovering unknown genes that utilize alternative decoding, as well as for analyzing frameshift sites. It is freely accessible at http://wilab.inha.ac.kr/FSFinder/.     

Aminopeptidase from Sphingomonas capsulata

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min(-1) for glutamate. At pH 7.5 (pH optimum) and 25 degrees C, the kinetic parameters for alanine para-nitroanilide were found to be k(cat) = 7600 min(-1) and K(m) = 14 mm. For alanine beta-naphthylamide, they were k(cat) = 860 min(-1) and K(m) = 6.7 mm. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.     

Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification

Recombinant Chinese hamster ovary (CHO) parental clones expressing a humanized antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy chain (HC) and light chain (LC) cDNA expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 representative parental clones were subjected to stepwise selection for increasing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 microM, their clonal variations in regard to antibody expression were found to be significant. Among 23 parental clones, only one clone (hu17) showed the significant increment of specific antibody productivity (q(Ab)) with increasing MTX concentration up to 0.32 microM. Compared with the parental clone (hu17), the q(Ab) of hu17 resistant at 0.32 microM MTX (hu17-0.32) was enhanced approximately 12.5-fold. To clarify the reason for the occurrence of clonal variations, Southern blot analyses of chromosomal DNAs derived from each amplified clone at 0.32 microM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it had only one 49-kb amplification unit including the LC and HC cDNAs. A fluorescent MTX competition assay showed that the resistance against MTX toxicity of the other clones without enhanced q(Ab) at 0.32 microM MTX was obtained by mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expression are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepwise selection.     

Modulation of the peptide-binding specificity of a single-chain class II major histocompatibility complex

We designed and expressed a single-chain class II major histocompatibility complex molecule capable of forming a stable complex with an antigenic peptide. The peptide-binding preference of the single-chain (sc) human leukocyte antigen derived from DRB5(*)0101 (DR51) was determined to be similar to that of the authentic one, which requires a bulky hydrophobic residue at position-1 (P1) as a primary anchor. For modulation of the peptide-binding affinity, we modified binding pocket 1 of sc DR51 by site-directed mutagenesis. The relative binding affinity of the engineered sc DR51 for several P1-substituted peptides was measured by competition assaying with a fluorescence labeled peptide. The sc DR51 molecule showed high affinity to the self-peptide derived from myelin basic protein, 87-98 with Phe as the P1 residue (F90F). While reduction of pocket 1 volume (betaG86V) decreased the affinity of F90F, it rather increased the affinity of the Ala-substituted peptide as to the P1 residue (F90A). Through more extensive engineering in the peptide-binding groove of the sc DR51 molecule, it is expected that we can construct sc DR51 variants with various peptide ligand motifs.     

Giant Ragweed Invasion is Not Well Controlled by Biotic Resistance

The effect of native plant restoration on invasion by giant ragweed (Ambrosia trifida), an invasive species, is currently unknown. We hypothesized that (1) functional group identity would be a good predictor of biotic resistance to A. trifida, and (2) mixtures of species would be more resistant to invasion than monocultures. Using seven functional traits, 37 native and non-native plants were divided into three functional groups that differed primarily in longevity and woodiness. We conducted a competition experiment using an additive competition design with A. trifida and monocultures or mixtures of 14 species. Biotic resistance was evaluated by calculating a relative competition index (RCI_avg) based on the average performance of A. trifida in treatments compared with that in control. In monocultures, RCI_avg of resident plants did not significantly differ among the three functional groups or within each functional group. The highest RCI_avg (40%) was observed for some fast-growing annuals (FG1) such as Zea mays and Secale cereal, which were strong competitors. RCI_avg of resident plants was not significantly greater in mixtures than in monocultures. Taken together, the results show that plant diversity did not control invasion by A. trifida and that giant ragweed invasion cannot be well controlled by biotic resistance.     

Management of invasive plants through ecological resistance

Despite debates on the real impact of plant invasion on native biodiversity, there remain many situations where exotic invasive plants must be managed and habitats restored. Restoration practices that build on plant community assembly principles could be useful to delay or prevent re-invasion after control, but there are still few syntheses of the biodiversity theory, ecological mechanisms and experimental evidence relevant to invasive plant management, possibly delaying applications. To provide such a synthesis, we review current knowledge on three key determinants of invasion success: biotic resistance, abiotic constraints, and propagule pressure. We elaborate on the ecological mechanisms at play for each determinant and emphasize, using case studies, their relevance for invasive plant management and ecological restoration. We find evidence that restoring a plant cover can enhance invasion resistance, but the challenge for both research and field applications is to understand how multiple determinants interact in relation to species traits in the fields. Failure to recognize these interactions and their effect on community assembly processes may explain some of the mixed species responses observed. While we need control and restoration case studies with local species at different sites, the development of a coherent, dynamic and adaptive framework around biotic/ecological resistance will have to go beyond the idiosyncrasy of the many species and systems being tested. Emphasizing the functional diversity of the restored community seems a promising approach when facing potentially multiple invaders and/or fluctuating abiotic conditions.     

Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol, a natural analog and a metabolite of resveratrol, against glutamate-mediated oxidative injury in HT22 neuronal cells

Neuronal cell death caused by oxidative stress is common in a variety of neural diseases and can be investigated in detail in cultured HT22 neuronal cells, where the amino acid glutamate at high concentrations causes glutathione depletion by inhibition of the glutamate/cystine antiporter system, intracellular accumulation of reactive oxygen species (ROS) and eventually oxidative stress-induced neuronal cell death. Using this paradigm, we have previously reported that resveratrol (3,5,4′-trans-trihydroxystilbene) protects HT22 neuronal cells from glutamate-induced oxidative stress by inducing heme oxygenase (HO)-1 expression. Piceatannol (3,5,4′,3′-trans-trihydroxystilbene), which is a hydroxylated resveratrol analog and one of the resveratrol metabolites, is estimated to exert neuroprotective effect similar to that of resveratrol. The aim of this study, thus, is to determine whether piceatannol, similarly to resveratrol, would protect HT22 neuronal cells from glutamate-induced oxidative stress. Glutamate at high concentrations induced neuronal cell death and ROS formation. Piceatannol reduced glutamate-induced cell death and ROS formation. The observed cytoprotective effect was much higher when HT22 neuronal cells were pretreated with piceatannol for 6 or 12 h prior to glutamate treatment than when pretreated for 0.5 h. Piceatannol also increased HO-1 expression and HO activity via its activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, neuroprotective effect of piceatannol was partly (but not completely) abolished by either down-regulation of HO-1 expression or blockage of HO-1 activity. Taken together, our results suggest that piceatannol, similar to resveratrol, is capable of protecting HT22 neuronal cells against glutamate-induced cell death, at least in part, by inducing Nrf2-dependent HO-1 expression.