SUMO1 promotes Aβ production via the modulation of autophagy

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-β (Aβ) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aβ levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aβ production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aβ was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aβ production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.     

Generation of transgenic silkworms for production of erythropoietin in Bombyx mori

There have been many attempts to generate various essential proteins using transformed E. coli systems. However, prokaryote systems are not equipped with the protein maturation mechanisms necessary to generate eukaryotic proteins. In this sense, among the eukaryotes, silkworms have major merits in overcoming the difficulties. Such protein maturation mechanisms are available in silkworms. In this study, a transgenic silkworm producing rhEPO in the cocoon was generated and purified. Specifically, we constructed a transgenic silkworm using a vector system that could be controlled to the next generation. To accomplish this, we microinjected the system into eggs laid during the preblastoderm stage. The rhEPO was then purified from transgenic silkworm cocoons using a Con A affinity column. The biological activity of rhEPO isolated from the cocoon of transgenic silkworms was then assessed in a cell culture system using an EPO-dependent cell line, F-36E. Next, PCR analysis was used to demonstrate that stable gene expression can occur in the embryos of the silkworm, Bombyx. mori. Transgenic silkworms were then selected and observed to ensure that the transgenic silkworm was maintained and transmitted to their progeny. The rhEPO was subsequently purified from the transgenic silkworm cocoon and the electrophoretic pattern of the purified rhEPO revealed a protein band with a molecular weight of approximately 20 kDa. A total of 3 mg of rhEPO was eluted from 10 g of cocoons. The proliferation of F36E cells for 25 ng/ml rhEPO was 1.32, while the proliferation for 2.5 IU/ml hEPO was 1.32. The proliferation of these cells could be induced by commercial hEPO, as well as by rhEPO from transgenic silkworm cocoons. An in vivo test of mice treated with rhEPO revealed relatively high RBC values when compared to normal mice. These results indicated that purified glycosylated EPO from transgenic silkworms had biological activities. Overall, the transgenic silkworm technique will be very useful for the large scale production of proteins for diagnostic and therapeutic purposes.     

Association between polymorphisms of Myf5 and POU1F1 genes with growth and carcass traits in Hanwoo (Korean cattle)

Myogenic factor 5 (Myf5) and POU class 1 homeobox 1 (POU1F1) genes play important roles in growth and development of mammals. Bovine Myf5 and POU1F1 were characterized to detect genetic variation at these loci and to replace them to economic traits in 367 cattle representing Hanwoo (325) and Angus (37). Two single nucleotide polymorphisms (SNPs) were identified in intron 2 (A1948G SNP) of Myf5 and exon 6 (A15635G SNP) of POU1F1 by sequence analyses of genomic DNA. Statistical analysis indicated that the Myf5 polymorphisms significantly (0.05) associated backfat thickness and live weight at 6-months-of-age and that POU1F1 polymorphisms significantly influenced carcass weight and live weight at 24-month of age, and backfat thickness. The interaction between Myf5 and POU1F1 was significant on carcass weight, M. longissimus dorsi area, backfat thickness and marbling score. The results implicate Myf5 and POU1F1 as candidate genes of growth and carcass traits, and suggest that the interaction between Myf5 and POU1F1 strongly affect growth and carcass traits in cattle.     

Molecular cloning and characterization of PLCB1 (phospholipase C, beta 1) gene from the olive flounder, Paralichthys olivaceus

PLCB1 (phospholipase C, beta 1) cDNA was cloned from olive flounder (Paralichthys olivaceus) cDNA via rapid amplification of cDNA ends (RACE). The cDNA for olive flounder PLCB1 (PoPLCB1) encodes for a polypeptide of 1,244 amino acids in length containing a well-conserved PH domain, catalytic X and Y domains, a C2 domain. From the sequence information of the BAC library, we assembled a contig containing the whole flounder PLCB1 cDNA sequences, and determined the exon/intron structure of the gene spanning > 110,743 bp DNA. PoPLCB1 gDNA sequences demonstrated the new sequence (exon 15), which has only been observed in the fish, is located between the X and Y domain of the PLCB1, and that PoPLCB1 exists as two splice variants-PoPLCB1a (1,244 amino acids) and PoPLCB1b (1,210 amino acids). Phylogenic analysis and sequence comparison of PoPLCB1 with other PLC isozymes showed a close relationship with the PLCB1 isozyme. Tissue-specific mRNA of PoPLCB1 was expressed predominantly in the brain and heart tissues. Between the two splicing variants of PoPLB1 in RT-PCR by tissue, PoPLCB1a showed a major expression pattern in more diverse types of tissues than the PoPLCB1b. PoPLCB1 gene expression was compared with that of the inflammatory cytokines IL-1β and TNF-α in infected spleen and kidney tissues via real-time RT-PCR assays following stimulation with LPS. After the stimulation, the expression of PoPLCB1 increased significantly prior to IL-1B and TNF-α expression. This provided direct evidence suggesting that PoPLCB1 may perform a crucial role in immune responses against pathogens and in inflammation.     

New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO₂. A unique [NiFe₄S₄] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH•Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe₃S₄] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.     

The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways.

Here we characterized a rice (Oryza sativa L.) blast lesion mimic (blm) mutant, identified previously in an N-methyl-N-nitrosourea-mutagenized population of the cultivar Hwacheong (wild type). The rice blm displayed spontaneous necrotic lesion formation on the leaves during development under long-day condition and temperature shift from 28 to 24 degrees C in the absence of obvious stress/disease, and provided us with a highly reproducible and convenient experimental system in the growth chamber to study blm. The blm phenotype resembled to the cell death of hypersensitive reaction (HR), and subsequent, two-dimensional gel electrophoresis (2-DGE) revealed induction of many leaf proteins; prominent among them were the three pathogenesis-related (PR) marker proteins of class 5 (one spot) and 10 (two spots). Interestingly, the rice blm manifested HR against all races tested of the rice blast fungus (Magnaporthe grisea), providing high resistance in a non-race specific manner. It was also observed that blm was highly resistant to hydrogen peroxide treatment. Using 2-DGE immunoblotting, we identified the presence of 4 new spots cross-reacting with a superoxide dismutase (SOD) antibody, only in blm, suggesting the expression of potentially new SOD protein (isoforms) during lesion formation. In the leaves of blm, autofluorescent compounds accumulated in and around the site of lesion progression. Moreover, enhanced levels of two major rice phytoalexins, sakuranetin and momilactone A were also observed in the leaves of blm. These results indicate that blm confers broad-spectrum resistance to multiple pathogens, and so, it could be hypothesized that the BLM gene product may control the HR-like cell death and its associated multiple defense signaling pathways, as evidenced by induction of known hallmark features (proteins/metabolites) linked with the defense responses, in rice.