Complete Chloroplast Genome Sequence of <Emphasis Type="Italic">Glycine max</Emphasis> and Comparative Analyses with other Legume Genomes

Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.     

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

Chickpea (Cicerarietinum, Leguminosae), an important grain legume, is widely used for food and fodder throughout the world. We sequenced the complete plastid genome of chickpea, which is 125,319bp in size, and contains only one copy of the inverted repeat (IR). The genome encodes 108 genes, including 4 rRNAs, 29 tRNAs, and 75 proteins. The genes rps16, infA, and ycf4 are absent in the chickpea plastid genome, and ndhB has an internal stop codon in the 5'exon, similar to other legumes. Two genes have lost their introns, one in the 3'exon of the transpliced gene rps12, and the one between exons 1 and 2 of clpP; this represents the first documented case of the loss of introns from both of these genes in the same plastid genome. An extensive phylogenetic survey of these intron losses was performed on 302 taxa across legumes and the related family Polygalaceae. The clpP intron has been lost exclusively in taxa from the temperate "IR-lacking clade" (IRLC), whereas the rps12 intron has been lost in most members of the IRLC (with the exception of Wisteria, Callerya, Afgekia, and certain species of Millettia, which represent the earliest diverging lineages of this clade), and in the tribe Desmodieae, which is closely related to the tribes Phaseoleae and Psoraleeae. Data provided here suggest that the loss of the rps12 intron occurred after the loss of the IR. The two new genomic changes identified in the present study provide additional support of the monophyly of the IR-loss clade, and resolution of the pattern of the earliest-branching lineages in this clade. The availability of the complete chickpea plastid genome sequence also provides valuable information on intergenic spacer regions among legumes and endogenous regulatory sequences for plastid genetic engineering.     

The N-terminal alanine-extended GLP-1/IgG-Fc fusion protein confers resistance to DPP-IV and reduces serum glucose level in db/db mice

The aim of this study was to develop novel long-acting glucagon-like peptide 1 (GLP-1) analogs resistant to dipeptidyl peptidase-IV (DPP-IV). We constructed three fusion proteins comprising GLP-1 and the human immunoglobulin gamma heavy chain (IgG-Fc); wild-type GLP-1 and IgG-Fc (GLP-1/IgG-Fc) and two N-terminal-extended fusion proteins in which an additional Ala (A) or Gly (G) was located on the N-terminus of GLP-1 (A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc). The fusion proteins expressed in CHO-K1 cells were secreted into medium and purified by Protein A affinity chromatography. Here, we show that the Ala or Gly-extended GLP-1/IgG-Fc fusion protein is resistant to DPP-IV and has increased half-life in vivo. To our surprise, the A-GLP-1/IgG-Fc fusion protein was more effective than wildtype GLP-1/IgG-Fc fusion protein in reducing blood glucose levels in db/db mice. Our findings suggest that the A-GLP-1/IgG-Fc fusion protein could be a potential long-acting GLP-1 receptor agonist for the treatment of insulin-resistant type 2 diabetes.     

Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts

Monellin is a naturally sweet protein that consists of two polypeptide chains and has potential uses as a highly potent non-carbohydrate sweetener. We aimed to make this protein more usable by increasing its stability and expressing it in a high-yielding system. MNEI is a modified version of the protein that consists of the two natural chains of monellin joined via a dipeptide linkage. In the thermostability analysis of MNEI variants, four mutated MNEIs, MNEI-E24L, MNEI-E24F, MNEI-E24W, and MNEI-E24A, had higher melting temperatures than wild-type MNEI and retained their sweet flavor even at temperatures above 70?°C. Our findings indicate that the increased stability of monellin allows it to retain its strong sweetness even under extreme conditions. We successfully overexpressed the thermostable MNEI mutants in tobacco chloroplasts. Here, we report that the MNEI mutants showed enhanced thermostability, and the stable forms of MNEI can be produced through plastid transformation in tobacco.     

Prognostic significance of sealed-off perforation in colon cancer: a prospective cohort study

Background

Perforated colon cancer is a rare complication, but has a high risk of recurrence. However, most studies have not distinguished sealed-off perforation from free perforation, and the prognosis is unclear. The aim of this study was to evaluate the oncologic outcome of colon cancer with sealed-off perforation.

Methods

Eighty-six consecutive patients who underwent resection for colon cancer with sealed-off or free perforation were included. We defined sealed-off perforation as a colon perforation with localized abscess identified on operative, computed tomography, or pathologic findings, with no evidence of free perforation, including fecal contamination and dirty fluid collection in the peritoneal cavity. Oncologic outcomes were compared between patients with colon cancer with sealed-off perforation and free perforation using a log-rank test and Cox regression analysis.

Results

The sealed-off perforation group included 62 patients, and 24 patients were in the free perforation group. TNM stage and lymphatic, venous, and perineural invasion were similar between the groups. The median follow-up period was 28.9?months (range 0–159). The sealed-off perforation group had better prognosis compared with the free perforation group in terms of progression-free survival (PFS) and overall survival (OS), although there were no statistically significant differences in PFS (5-year PFS 53.7% vs. 40.5%, p?=?0.148; 5-year OS 53.6% vs. 22.9%, p?=?0.001). However, in multivariable analysis using the Cox progression test, sealed-off perforation did not show a significant effect on cancer progression (p =?0.138) and OS (p =?0.727).

Conclusions

Colon cancer with sealed-off perforation showed no difference in prognosis compared with free perforation.

     

Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids

Background  

The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids.     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.