Expression and production of recombinant human interleukin-2 in potato plants

Interleukin-2 is a pharmacologically important cytokine secreted by T lymphocytes. Recombinant interleukin-2 has been produced and found to be useful for many medical applications. Mass production of recombinant interleukin-2 will be prerequisite for a wider application of this molecule. In this study we investigated the possibility of using potato tubers for the production of recombinant human interleukin-2 in large quantity. A binary vector carrying the human interleukin-2 gene under a potato tuber-specific promoter (patatin promoter) was constructed. Several potato transformants expressing the human interleukin-2 gene were generated by Agrobacterium-mediated transformation. Expression of the human interleukin-2 gene was confirmed by Northern blotting and the protein level was determined by Western blot analyses. A bioassay revealed that human interleukin-2 expressed in the potato tuber supported proliferation of interleukin-2-dependent cells, CTLL-2. We found that the recombinant protein in the 2-week-old microtuber has the highest activity (115 units per gram of microtuber) and estimated that an average yield for a potato (average 200 g per potato) was 23,000 units of rhIL-2 activity. The results suggest that the potato tuber is an excellent system for the mass production of biologically active human interleukin-2.     

Expression and regulation of theRSI-1 gene during lateral root initiation

The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots. Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process.     

Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a β-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (α/α)₆ fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.     

1H, 13C and 15N chemical shift assignments of Ninjurin1 Extracellular N-terminal Domain

Cell adhesion molecules play a crucial role in fundamental biological processes via regulating cell–cell interactions. Nerve injury induced protein1 (Ninjurin1) is a novel adhesion protein that has no significant homology with other known cell adhesion molecules. Here we present the assignment of an 81 aa construct for human Ninjurin1 Extracellular N-Terminal (ENT) domain, which comprises the critical adhesion domain.     

ABCA3 is critical for lamellar body biogenesis in vivo

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3-/- knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/-, neonates survived. The lungs from E18.5 and E19.5 Abca3-/- mice were less mature than wild type. Alveolar type 2 cells from Abca3-/- embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/- compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3-/- embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation.     

New Record of Feather Mite,Neopteronyssus bilineatus Mironov, 2003 (Arachnida: Pteronyssidae), from a Grey-Capped Pygmy Woodpecker,Yungipicus canicapillus in Republic of Korea

This study intended to record a species of feather mite, Neopteronyssus bilineatus Mironov, 2003, (Arachnida: Pteronyssidae), from a grey-capped pygmy woodpecker, Yungipicus canicapillus (Blyth, 1845), in the Republic of Korea. Mite samples were collected from the flight feathers of a woodpecker, preserved directly in 95% ethyl alcohol, and then observed by a light microscope after specimen preparation. Morphology of Neopteronyssus bilineatus is distinguished from other pici group species by opisthosoma part with 2 longitudinal bends, tarsal seta rIII 3 times longer than tarsus III in males, and 2 elongated hysteronotal plates extending beyond the level of setae e2 in females. In the present study, a species of feather mite, N. bilineatus, was newly recorded from Y. canicapillus in Korean fauna.     

Association of GNLY Genetic Polymorphisms with Chronic Liver Disease in a Korean Population

Granulysin (GNLY) is found in cytotoxic granules of cytolytic T lymphocytes and natural killer (NK) cells, which are critical for hepatitis B virus (HBV) clearance. GNLY cytotoxicity plays an important role in the defense against viruses or intracellular bacteria. We hypothesized that genetic variation in the GNLY gene could affect the resistance of hosts against HBV infection. We compared the distribution frequencies of GNLY polymorphisms between an HBV-induced chronic liver disease (CLD) group and a spontaneous recovery (SR) control group to determine whether GNLY polymorphisms play a role in HBV clearance. A total of 117 patients in the SR group and 230 patients in the CLD group were enrolled. Samples derived from complex infections, including hepatitis C and human immunodeficiency virus, and those associated with insufficient clinical information (10 samples in SR and 24 samples in CLD) were excluded from the study. The final analysis included 107 SR and 206 CLD samples. DNA was extracted from peripheral blood, and GNLY genotypes were determined by the GoldenGate(?) method. The genotype distribution of the single-nucleotide polymorphisms (SNPs) rs2886767 (C>T), rs1561285 (G>C), and rs11127 (T>C) were significantly different between the SR and CLD groups in a recessive model (p<0.015). These three SNPs were in a complete linkage disequilibrium (LD) block. Diplotype distributions of haplotype (HT) 1 (C-G-T) and HT2 (T-C-C) were significantly different between the SR and CLD groups in a recessive model (p=0.025) and a dominant model (p=0.008). All p-values remained significant after multiple comparisons. GNLY polymorphism genotypes and diplotypes were associated with the chronicity of HBV. These data suggested that genetic variation of GNLY may be an important factor in HBV clearance through the CD8+ T or NK cell-mediated removal of HBV-infected cells from the host.     

Urokinase-Type Plasminogen Activator Induces BV-2 Microglial Cell Migration Through Activation of Matrix Metalloproteinase-9

In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.