Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis / 生物工程学报

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

Genetic analysis of a rare fetus with mandibulofacial dysostosis Guion-Almeida type / 中华医学遗传学杂志

OBJECTIVE@#To delineate the clinical and genetic features of a fetus with micrognathia, low-set ears, microtia, polyhydramnios and anechoic stomach by ultrasonography.@*METHODS@#Whole exome sequencing (WES) was carried out to detect genetic variant in the fetus, for which routine chromosomal karyotyping and chromosomal microarray analysis (CMA) yielded no positive finding. Candidate variants were verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#WES revealed that the fetus has carried a de novo nonsense c.2302C>T (p.Q768X) variant in exon 23 of the EFTUD2 gene, which was detected in neither parent. The variant was unreported previously and may lead to premature termination of the translation of EFTUD2 protein at the 768th amino acid. Bioinformatic analysis predicted the amino acid to be highly conserved and may alter the structure and function of the EFTUD2 protein.@*CONCLUSION@#The c.2302C>T variant of the EFTUD2 gene probably underlay the mandibulofacial dysostosis Guion-Almeida type in the fetus. Discovery of the novel variant has enriched variant spectrum of the EFTUD2 gene and provided a basis for genetic counseling and prenatal diagnosis for the family.

Fungal Diversity and Enzyme Activity Associated with the Macroalgae, Agarum clathratum

Agarum clathratum, a brown macroalgae species, has recently become a serious environmental problem on the coasts of Korea. In an effort to solve this problem, fungal diversity associated with decaying A. clathratum was investigated and related β-glucosidase and endoglucanase activities were described. A total of 233 fungal strains were isolated from A. clathratum at 15 sites and identified 89 species based on morphology and a multigene analysis using the internal transcribed spacer region (ITS) and protein-coding genes including actin (act), β-tubulin (benA), calmodulin (CaM), and translation elongation factor (tef1). Acremonium, Corollospora, and Penicillium were the dominant genera, and Acremonium fuci and Corollospora gracilis were the dominant species. Fifty-one species exhibited cellulase activity, with A. fuci, Alfaria terrestris, Hypoxylon perforatum, P. madriti, and Pleosporales sp. Five showing the highest enzyme activities. Further enzyme quantification confirmed that these species had higher cellulase activity than P. crysogenum, a fungal species described in previous studies. This study lays the groundwork for bioremediation using fungi to remove decaying seaweed from populated areas and provides important background for potential industrial applications of environmentally friendly processes.

New Report of Three Unrecorded Species in Trichoderma harzianum Species Complex in Korea

The genus Trichoderma (Hypocreaceae, Ascomycota) consists of globally distributed fungi. Among them, T. harzianum, one of the most commonly collected Trichoderma species, had been known as a polyphyletic or aggregate species. However, a total of 19 species were determined from the polyphyletic groups of T. harzianum. Thus, we explored Korean “T. harzianum” specimens that were collected in 2013–2014. These specimens were re-examined based on a recent study with translate elongation factor 1-alpha (EF1α) sequences to reveal cryptic Trichoderma species in Korea. As a result, four different species, T. afroharzianum, T. atrobruneum, T. pyramidale, and T. harzianum, were identified. Except T. harzianum, the other three species have not been reported in Korea. In this work, we describe these species and provide figures.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4–11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease β subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Identification and Characterization of Pseudocercospora pyricola Causing Leaf Spots on Aronia melanocarpa

Leaf spot disease on black chokeberry (Aronia melanocarpa) was observed at several locations in Korea during 2014–2015. Leaf spots were distinct, scattered over the leaf surface and along the leaf border, subcircular to irregular and brown surrounded by a distinct dark color, and were expanded and coalesced into irregularly shaped lesions. Severely infected leaves became dry and fell off eventually. The causative agent was identified as Pseudocercospora pyricola. Morphological observations and phylogenetic analyses of multiple genes, including internal transcribed spacer, translation elongation factor 1-alpha, actin, and the large subunit ribosomal DNA were conducted. The pathogenicity test was conducted twice yielding similar results, fulfilling Koch's postulates. To our knowledge, this is the first report on P. pyricola infection of A. melanocarpa globally.

First Report of Dieback Caused by Lasiodiplodia theobromae in Strawberry Plants in Korea

Dieback in strawberry (Seolhyang cultivar) was first observed during the nursery season (June to September) in the Nonsan area of Korea in the years 2012 and 2013. Initial disease symptoms included dieback on runners, as well as black rot on roots, followed by wilting and eventually blackened, necrotic discoloration in the crowns of daughter plants. A fungus isolated from the diseased roots, runners, and crowns is close to Lasiodiplodia theobromae based on morphological characteristics. Analysis of a combined dataset assembled from sequences of the internal transcribed spacer and translation elongation factor 1-alpha genes grouped nine fungal isolates with the type strain of L. theobromae. The isolates showed strong pathogenicity on strawberry cultivars Kumhyang, Seolhyang, and Akihimae, fulfilling Koch's postulates. Based on these results, the pathogen responsible for dieback on strawberry plants in Korea was identified as L. theobromae.

The Level of Autoantibodies Targeting Eukaryote Translation Elongation Factor 1 α1 and Ubiquitin-Conjugating Enzyme 2L3 in Nondiabetic Young Adults

BACKGROUND: The prevalence of novel type 1 diabetes mellitus (T1DM) antibodies targeting eukaryote translation elongation factor 1 alpha 1 autoantibody (EEF1A1-AAb) and ubiquitin-conjugating enzyme 2L3 autoantibody (UBE2L3-AAb) has been shown to be negatively correlated with age in T1DM subjects. Therefore, we aimed to investigate whether age affects the levels of these two antibodies in nondiabetic subjects. METHODS: EEF1A1-AAb and UBE2L3-AAb levels in nondiabetic control subjects (n=150) and T1DM subjects (n=101) in various ranges of age (18 to 69 years) were measured using an enzyme-linked immunosorbent assay. The cutoff point for the presence of each autoantibody was determined based on control subjects using the formula: [mean absorbance+3×standard deviation]. RESULTS: In nondiabetic subjects, there were no significant correlations between age and EEF1A1-AAb and UBE2L3-AAb levels. However, there was wide variation in EEF1A1-AAb and UBE2L3-AAb levels among control subjects <40 years old; the prevalence of both EEF1A1-AAb and UBE2L3-AAb in these subjects was 4.4%. When using cutoff points determined from the control subjects <40 years old, the prevalence of both autoantibodies in T1DM subjects was decreased (EEFA1-AAb, 15.8% to 8.9%; UBE2L3-AAb, 10.9% to 7.9%) when compared to the prevalence using the cutoff derived from the totals for control subjects. CONCLUSION: There was no association between age and EEF1A1-AAb or UBE2L3-AAb levels in nondiabetic subjects. However, the wide variation in EEF1A1-AAb and UBE2L3-AAb levels apparent among the control subjects <40 years old should be taken into consideration when determining the cutoff reference range for the diagnosis of T1DM.

First Report on Fusarium Wilt of Zucchini Caused by Fusarium oxysporum, in Korea

Fusarium wilt of zucchini in Jeonju, Korea, was first noticed in May 2013. Symptoms included wilting of the foliage, drying and withering of older leaves, and stunting of plants. Infected plants eventually died during growth. Based on morphological characteristics and phylogenetic analyses of the molecular markers (internal transcribed spacer rDNA and translation elongation factor 1alpha), the fungus was identified as Fusarium oxysporum. Pathogenicity of a representative isolate was demonstrated via artificial inoculation, and it satisfied Koch's postulates. To our knowledge, this is the first report of F. oxysporum causing wilt of zucchini in Korea.

First Report of Fusarium subglutinans Causing Leaf Spot Disease on Cymbidium Orchids in Korea

In 2006~2010, leaf spot symptoms, that is, small, yellow spots that turned into dark brown-to-black lesions surrounded by a yellow halo, were observed on Cymbidium spp. in Gongju, Taean, and Gapyeong in Korea. A Fusarium species was continuously isolated from symptomatic leaves; in pathogenicity testing, isolates caused leaf spot symptoms consisting of sunken, dark brown lesions similar to the original ones. The causal pathogen was identified as Fusarium subglutinans based on morphological and translation elongation factor 1-alpha sequence analyses. This is the first report of F. subglutinans as the cause of leaf spot disease in Cymbidium spp. in Korea.

Cladosporium cladosporioides and C. tenuissimum Cause Blossom Blight in Strawberry in Korea

Blossom blight in strawberry was first observed in a green house in Nonsan, Damyang, and Geochang areas of Korea, between early January to April of 2012. Disease symptoms started as a grey fungus formed on the stigma, which led to the blossom blight and eventually to black rot and necrosis of the entire flower. We isolated the fungi purely from the infected pistils and maintained them on potato dextrose agar (PDA) slants. To test Koch's postulates, we inoculated the fungi and found that all of the isolates caused disease symptoms in the flower of strawberry cultivars (Seolhyang, Maehyang, and Kumhyang). The isolates on PDA had a velvet-like appearance, and their color ranged between olivaceous-brown and smoky-grey to olive and almost black. The intercalary conidia of the isolates were elliptical to limoniform, with sizes ranging from 5.0~10.5 x 2.5~3.0 microm to 4.0~7.5 x 2.0~3.0 microm, respectively. The secondary ramoconidia of these isolates were 0- or 1-septate, with sizes ranging betweem 10.0~15.0 x 2.5~3.7 microm and 8.7~11.2 x 2.5~3.2 microm, respectively. A combined sequence analysis of the internal transcribed spacer regions, partial actin (ACT), and translation elongation factor 1-alpha (TEF) genes revealed that the strawberry isolates belonged to two groups of authentic strains, Cladosporium cladosporioides and C. tenuissimum. Based on these results, we identified the pathogens causing blossom blight in strawberries in Korea as being C. cladosporioides and C. tenuissimum.

First Report on Isolation of Penicillium adametzioides and Purpureocillium lilacinum from Decayed Fruit of Cheongsoo Grapes in Korea

Two species, Penicillium adametzioides and Purpureocillium lilacinum, were isolated from decayed grapes (cv. Cheongsoo) in Korea. Each species was initially identified by phylogenetic analysis of a combined dataset of two genes. Internal transcribed spacer (ITS) and beta-tubulin (BT2) genes were used for identification of Penicillium adametzioides, and ITS and partial translation elongation factor 1-alpha (TEF) genes were used for identification of Purpureocillium lilacinum. Morphologically, they were found to be identical to previous descriptions. The two species presented here have not been previously reported in Korea.

Molecular and Morphological Characterization of Endophytic Heterobasidion araucariae from Roots of Capsicum annuum L. in Korea

A species of Heterobasidion was encountered during a diversity study of endophytic fungi from healthy root tissues of chili pepper (Capsicum annuum L.) in Korea. The fungal species (CNU081069) was identified as Heterobasidion araucariae based on phylogenetic analyses of the internal transcribed spacer and translation elongation factor gene sequences. Morphological descriptions of the endophytic isolate matched well with the previous references and supported the molecular identification. The fungus Heterobasidion araucariae CNU081069 is new to Korea.

The Role of Protein Phosphorylation in the Gustatory Cortex and Amygdala During Taste Learning

Protein phosphorylation and dephosphorylation form a major post-translation mechanism that enables a given cell to respond to ever-changing internal and external environments. Neurons, similarly to any other cells, use protein phosphorylation/dephosphorylation to maintain an internal homeostasis, but they also use it for updating the state of synaptic and intrinsic properties, following activation by neurotransmitters and growth factors. In the present review we focus on the roles of several families of kinases, phosphatases, and other synaptic-plasticity-related proteins, which activate membrane receptors and various intracellular signals to promote transcription, translation and protein degradation, and to regulate the appropriate cellular proteomes required for taste memory acquisition, consolidation and maintenance. Attention is especially focused on the protein phosphorylation state in two forebrain areas that are necessary for taste-memory learning and retrieval: the insular cortex and the amygdala. The various temporal phases of taste learning require the activation of appropriate waves of biochemical signals. These include: extracellular signal regulated kinase I and II (ERKI/II) signal transduction pathways; Ca(2+)-dependent pathways; tyrosine kinase/phosphatase-dependent pathways; brain-derived neurotrophicfactor (BDNF)-dependent pathways; cAMP-responsive element bindingprotein (CREB); and translation-regulation factors, such as initiation and elongation factors, and the mammalian target of rapamycin (mTOR). Interestingly, coding of hedonic and aversive taste information in the forebrain requires activation of different signal transduction pathways.

A análise comparativa entre alongamento muscular sob eletroestimulação e inibição ativa em atletas amadores de futebol / Analysis comparative between muscles strenghtening electric stimulation and active inhibition in soccer athlethes

Na pesquisa foram utilizados duas técnicas de alongamento, a eletroestimulação e a inibição ativa, onde através dos resultados obtidos procurou verificar a eficácia das mesmas. Foram estudados 15 atletas da seleção universitária de futebol, divididos igualmente em 3 grupos, com idade entre 18 e 25 anos. O estudo foi aplicado em 3 etapas: grupo I, eletroestimulação com 10 Hz de freqüência, 250us de largura de pulso, durante 10 minutos, através de dois eletrodos instalados sobre o ventre muscular dos ísquios-tibiais; grupo II, inibição ativa com contração muscular dos ísquios-tibiais por 10s seguido por um alongamento mantido por 30s, durante 10 minutos; grupo III, para controle, no qual os voluntários permaneceram em decúbito dorsal durante 10 minutos. Os atletas foram avaliados através da goniometria inicial e final do quadril com joelho em extensão total. A análise dos resultados mostrou que nas comparações entre os instantes inicial e final, houve variação significativa nos grupos submetidos aos protocolos I e II; no grupo controle não houve variação, e ambos os grupos de forma superior ao grupo controle. na comparação entre grupos, a técnica de inibição ativa mostrou-se mais eficaz em relação ao grupo de eletroestimulação e ambos ao grupo controle. Conclui-se que as técnicas aplicadas foram eficientes no aumento do grau de amplitude articular dos atletas; contudo a técnica de inibição ativa foi mais eficaz.

Comparison of Protein Expression in Normal Myometrium and Uterine Leiomyoma Using Two-Dimensional Gel Electrophoresis in Korean Women / 대한산부인과학회잡지

OBJECTIVE: Comparison of protein expression by two-dimensional gel electrophoresis (2-DE) in normal myometrium and uterine leiomyoma in Korean women. METHODS: Normal myometrium and uterine leiomyoma tissues were solubilized with 2-DE buffer and the first dimension of PROTEAN IEF CELL, isoelectric focusing (IEF), was performed using pH4-8 linear IPG strips of 17 cm. And then running 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and sliver stain. Scanned image analyzed using PDQuest 2-D softwareTM. Protein spot spectrum was identified by assisted laser desorption/ionization-time of fighting (MALDI-TOF) and the protein mass spectrums identification were performed by searching protein databases of Swiss-prot/TrEMBL, Mascot and MS-FIT. RESULTS: In this study, we found 17 up-regulated proteins (phosphate carrier protein, 60 kDa heat shock protein, acidic calcium-independent, glutathione transferase omega, chloride intracellular channel 4, Ras-related protein Rab-11B, phosphatidylinositol transfer protein alpha isoform, type II keratin subunit protein, Cofilin 2 isoform 1, transgelin, ATP carrier protein, alpha-catenin homolog, parkinson disease 2, apo-cellular retinoic acid binding protein II, osteoglycin preproprotein, proteasome activator subunit 1 isoform, Unnamed protein) and 7 down-regulated proteins (Serum amyloid P component, annexin IV, alpha 1 actin precursor, hypoxanthine-guanine phosphoribosyltransferase, tumor necrosis factor receptor superfamily member EDAR precursor, peroxiredoxin 2, translation elongation factor EF-Tu precursor) between myometrium and leiomyoma. CONCLUSION: 2-DE offer total protein expression between normal myometrium and uterine leiomyoma, and searching of differently expressed protein for the diagnostic markers of leiomyoma.

Detection of antibodies against glucose 6-phosphate isomerase in synovial fluid of rheumatoid arthritis using surface plasmon resonance (BIAcore)

We have used a surface plasmon resonance biosensor (SPR, BIACORE 2000) to detect antibodies against glucose 6-phosphate isomerase (GPI) in synovial fluids of rheumatoid arthritis (RA) and osteoarthritis (OA). Recombinant human GPI proteins fused with or without NusA were expressed in E. coli, purified to homogeneity and immobilized in flow cells of CM5 sensor chips. The flow cells immobilized with NusA protein or bovine serum albumin were used to monitor non-specific binding. Synovial fluid samples from RA patients showed a significantly higher level of binding to recombinant GPI proteins than samples from OA patients. Proteins which bound to the recombinant GPI proteins were confirmed to be immunoglobulin through the administration of anti-human immunoglobulin. NusA fusion protein was excellent for this assay because of a low background binding activity in the SPR analysis and its advantage of increased solubility in recombinant protein production. These results suggested a useful utilization of recombinant NusA-GPI fusion protein for the detection of autoantibodies against GPI in RA patients.

Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.

Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits.

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.