Development of single-stranded DNA aptamers for specific Bisphenol a detection

The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 10(15) random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4'-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol-gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules.     

High-mobility group box 1 is essential for mitochondrial quality control

Mitochondria are organelles centrally important for bioenergetics as well as regulation of apoptotic death in eukaryotic cells. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromatin-associated protein which maintains nuclear homeostasis, is also a critical regulator of mitochondrial function and morphology. We show that heat shock protein beta-1 (HSPB1 or HSP27) is the downstream mediator of this effect. Disruption of the HSPB1 gene in embryonic fibroblasts with wild-type HMGB1 recapitulates the mitochondrial fragmentation, deficits in mitochondrial respiration, and adenosine triphosphate (ATP) synthesis observed with targeted deletion of HMGB1. Forced expression of HSPB1 reverses this phenotype in HMGB1 knockout cells. Mitochondrial effects mediated by HMGB1 regulation of HSPB1 expression serve as a defense against mitochondrial abnormality, enabling clearance and autophagy in the setting of cellular stress. Our findings reveal an essential role for HMGB1 in autophagic surveillance with important effects on mitochondrial quality control.     

Biosynthesis of a novel recombinant peptide derived from hPTH(1-34)

A recombinant human parathyroid hormone fragment, Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro (MW=4550 Da), was developed by substituting Arg for Leu at position 11 and adding Pro-Pro at the carboxyl terminus. Following a single injection (0.5-13.5 μg/bird) of the rhPTH fragment, the serum calcium level in chickens increased 12-42% (P<0.05) after 1h as determined by the Parson's Chicken Assay. The functional activity of Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro may be due to removal of the N-terminus Pro-Pro- by X-prolyl dipeptidyl peptidase IV (DPPIV) in vivo, increasing its activity compared to Pro-Pro-hPTH(1-34). This artificial rhPTH fragment could be used to increase calcium mobilization and potentially improve bone health.     

Enhanced production of tropane alkaloids in transgenic <Emphasis Type="Italic">Scopolia parviflora</Emphasis> hairy root cultures over-expressing putrescine <Emphasis Type="Italic">N</Emphasis>-methyl transferase (PMT) and hyoscyamine-6β-hydroxylase (H6H)

Scopolia parviflora adventitious roots were metabolically engineered by co-expression of the two gene putrescine N-methyl transferase (PMT) and hyoscyamine-6β-hydroxylase (H6H) cDNAs with the aid of Agrobacterium rhizogenes. The transformed roots developed into morphologically distinct S. parviflora PMT1 (SpPMT1), S. parviflora PMT1 (SpPMT2), and S. parviflora H6H (SpH6H) transgenic hairy root lines. Consequent to the introduction of these key enzyme genes, the production of the alkaloids hyoscyamine and scopolamine was enhanced. Among the transgenic hairy root lines, SpPMT2 line possessed the highest growth index. The treatment of transgenic hairy roots with growth regulators further enhanced the production of scopolamine. Thus, the results suggest that PMT1, PMT2, and H6H genes may not only be involved in the metabolic regulation of alkaloid production but also that these genes may play a role in the root development.     

Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.     

Peroxiredoxin II is an essential antioxidant enzyme that prevents the oxidative inactivation of VEGF receptor-2 in vascular endothelial cells

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.     

Functional analysis of a <Emphasis Type="Italic">Hansenula polymorpha MNN2-2</Emphasis> homologue encoding a putative UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine transporter localized in the endoplasmic reticulum

The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.