Amyloid beta-protein (Abeta)1-40 protects neurons from damage induced by Abeta1-42 in culture and in rat brain

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 microm, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.     

Characterization of a small cryptic plasmid,pHP51, from a Korean isolate of strain 51 of Helicobacter pylori

The nucleotide sequence of a 3955-bp Helicobacter pylori plasmid, pHP51 was determined, and two open reading frames, ORF1 and ORF2, were identified. The deduced amino acid sequence of ORF1 was highly conserved (87-89%) among plasmid replication initiation proteins, RepBs. The function of ORF2 was not assigned because it lacked known functional domains or sequence similarity with other known proteins, although it had a HPFXXGNG motif that was also found in the cAMP-induced filamentation (fic) gene. Three kinds of repeats were present on the plasmid outside of the ORFs, including the R1 and R2 repeats that are common in H. pylori plasmids. One 100-bp sequence detected in the noncoding region of pHP51 was highly similar to the genomic sequence of H. pylori 26695.     

Degradation of N-acylhomoserine lactones,the bacterial quorum-sensing molecules,by acylase

Porcine kidney acylase I was shown to be able to deacylate N-acylhomoserine lactones, a family of chemicals employed by Gram-negative bacteria as quorum-sensing molecules for cell population density-dependent growth (such as biofilm formation). The enzyme transformed both N-butyryl-and N-octanoyl-L-homoserine lactones into L-homoserine. An optimal pH of 10 at 23 degrees C and an optimal temperature of 76 degrees C at pH 9 were found for the enzyme in hydrolyzing N-butyryl-homoserine lactone. At pH 9 and 23 degrees C, the enzymatic catalysis had a K(m) of 81+/-3 mM and a k(cat) of 127+/-2 nmol min(-1) per mg. The enzyme was also shown to be able to reduce the biofilm growth in an aquarium water sample. Potential physiological significance and medicinal/industrial applications of the N-acylhomoserine lactone-degrading activity of acylase are discussed.     

PSEUDOVIEWER2: Visualization of RNA pseudoknots of any type

Visualizing RNA pseudoknot structures is computationally more difficult than depicting RNA secondary structures, because a drawing of a pseudoknot structure is a graph (and possibly a nonplanar graph) with inner cycles within the pseudoknot, and possibly outer cycles formed between the pseudoknot and other structural elements. We previously developed PSEUDOVIEWER for visualizing H-type pseudoknots. PSEUDOVIEWER2 improves on the first version in many ways: (i) PSEUDOVIEWER2 is more general because it can visualize a pseudoknot of any type, including H-type pseudoknots, as a planar graph; (ii) PSEUDOVIEWER2 computes a drawing of RNA structures much more efficiently and is an order of magnitude faster in actual running time; and (iii) PSEUDOVIEWER2 is a web-based application program. Experimental results demonstrate that PSEUDOVIEWER2 generates an aesthetically pleasing drawing of pseudoknots of any type and that the new representation offered by PSEUDOVIEWER2 ensures uniform and clear drawings, with no edge crossing, for all types of pseudoknots. The PSEUDOVIEWER2 algorithm is the first developed for the automatic drawing of RNA secondary structures, including pseudoknots of any type. PSEUDOVIEWER2 is accessible at http://wilab.inha.ac.kr/pseudoviewer2/.     

Cloning of ribosomal protein S6 kinase cDNA and its involvement in meiotic maturation in Rana dybowskii oocytes

Several protein kinases are involved in the meiotic maturation of frog oocytes in order to activate the maturation-promoting factor (MPF). Among these kinases, the 90 kDa ribosomal protein S6 kinase (p90Rsk or Rsk) is directly phosphorylated and activated by the mitogen-activated protein kinase (MAPK). During Xenopus oocyte maturation, the activation of Rsk closely parallels that of MAPK. Both enzymes are dephosphorylated when the cytostatic factor (CSF) disappears after fertilization. Therefore, Rsk seems to play an essential role in the activation of MPF. To evaluate it in other frog oocytes, we cloned and characterized Rsk cDNA in Rana dybowskii oocytes. The cloned Rana Rsk cDNA had 2,961 bp of nucleotides, which contained a complete single open-reading frame with ATG codon and polyadenylation signal. The deduced amino acid sequence of Rana Rsk is 733 amino acids with 83 kDa. Rana Rsk shows a high homology (about 88%) with Xenopus Rsk. It also had two well-conserved kinase domains with specific phosphorylation sites, which are known to be essential for the activation of Rsk. A Northern analysis showed that Rana Rsk mRNA was strongly expressed in ovary tissue, but weakly in other tissue. Rana Rsk protein is expressed with the pTYB1 vector and purified with the IMPACT-CN system. The purified Rana Rsk cross-reacted with Xenopus, a p90Rsk2 antiserum. Therefore, we examined the phosphorylation of Rana Rsk during Rana oocyte maturation. In P4-treated oocytes, Rana Rsk was phosphorylated about 6-9 h, which correlated well with the germinal vesicle breakdown of Rana oocytes. Therefore, it is likely that Rana Rsk plays an important role in the meiotic maturation of seasonal breeding animals.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

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.