Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis / 生物工程学报

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

Whole-Brain Mapping of Direct Inputs to and Axonal Projections from GABAergic Neurons in the Parafacial Zone / 神经科学通报·英文版

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.

Five New Records of Soil-Derived Trichoderma in Korea: T. albolutescens, T. asperelloides, T. orientale, T. spirale, and T. tomentosum

Despite the huge worldwide diversity of Trichoderma (Hypocreaceae, Ascomycota), only about 22 species have been reported in Korea. Thus, between 2013 and 2015, soil-derived Trichoderma spp. were isolated to reveal the diversity of Korean Trichoderma. Phylogenetic analysis of translation elongation factor 1 alpha gene was used for identification. Among the soil-derived Trichoderma, Trichoderma albolutescens, T. asperelloides, T. orientale, T. spirale, and T. tomentosum have not been previously reported in Korea. Thus, we report the five Trichoderma species as new in Korea with morphological descriptions and images.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

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 and Characterization of Pestalotiopsis ellipsospora Causing Canker on Acanthopanax divaricatus

Acanthopanax divaricatus, a member of the Araliaceae family, has been used as an invigorant in traditional Korean medicine. During disease monitoring, a stem with small, irregular, brown lesions was sampled at a farm in Cheonan in 2011. The symptoms seen were sunken cankers and reddish-brown needles on the infected twig. The isolated fungal colonies were whitish, having crenated edges and aerial mycelium on the surface, and with black gregarious fruiting bodies. The reverse plate was creamy white. Conidia were 17~22 x 3.5~4.2 microm, fusiform, 4-septate, and straight to slightly curved. The nucleotide sequence of the partial translation elongation factor 1 alpha gene of the fungal isolate, shares 99% sequence identity with that of known Pestalotiopsis ellipsospora. Based on the results of the morphological and molecular analyses, the fungal isolate was identified as P. ellipsospora. In Korea, this is the first report of canker on A. divaricatus.

Lethal effects of a Mexican Beauveria bassiana (Balsamo) strain against Meccus pallidipennis (Stal)

The entomopathogenic fungus Beauveria bassiana (Balsamo 1835) Vuillemin is an effective alternative control agent against some agricultural pests and biological vectors of important diseases such as Chagas disease. In this work we studied an isolate of Beauveria bassiana from of the town of San Antonio Rayón, Puebla, Mexico and its entomopathogenic effects on Meccus pallidipennis (Stal 1872). Phylogenetic analysis using molecular comparison of the ITS and EF1α genes, showed that the resulting cladogram places the BUAP 04 strain with a relationship closer to the AFAO 9-6 strain, within the diversity of the B. bassiana sensu lato group. Although there was the possibility that BUAP 04 strain was a direct descendant of strains used in campaigns of biologic control, molecular study allowed us to recognize that it was a different fungus due to numerous inserts. A strain isolated from a T. dimiata was evaluated for pathogenicity against another triatoma (Meccus pallidipennis) species obtaining an LC50 of 4.16 x 10(6) spores/mL, confirming that the BUAP 04 strain is virulent for M. pallidipennis and could be a good prospect for formulations to control M. pallidipennis.

Antifungal activity of metabolites of the endophytic fungus Trichoderma brevicompactum from garlic

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8SITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13-epoxy-Δ9-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µgmL-1. Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µgmL-1. However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µgmL-1). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Identifying interacting proteins of a Caenorhabditis elegans voltage-gated chloride channel CLH-1 using GFP-Trap and mass spectrometry / 生理学报

Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.

Effects of eEF1A1 re-expression on proliferation and apoptosis of Jurkat cells with knocked down eEF1A1 gene and its mechanisms / 中国实验血液学杂志

This study was aimed to explore the effects of expressing eukaryotic elongation factor 1A1 (eEF1A1) on proliferation and apoptosis in human acute T lymphocytic leukemia (T-ALL) cell line Jurkat with knocked down eEF1A1 gene and its mechanisms. eEF1A1-expressing lentivirus (LV) was constructed and used to transfect the Jurkat cells with knocked down eEF1A1 gene. Then, the expressions of eEF1A1 mRNA and protein were detected by real time PCR(RT-PCR) and Western blot respectively.Cell proliferation, apoptosis and cycle were detected by MTT method, Annexin V-APC labeling and DNA ploidy analysis respectively. The related protein expressions of phosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway were detected by Western blot. The results indicated that eEF1A1 mRNA and protein expressions of Jurkat cells with knocked down eEF1A1 gene were re-established by constructing eEF1A1-expression LV. Compared with negative control group (transfected with negative control LV and eEF1A1-shRNA LV), cell proliferation in eEF1A1 expression group was significantly enhanced, cell apoptosis was remarkably inhibited, percentage of cells in G0/G1 phase was significantly reduced alone with increased percentage of cells in S and G2/M phase, and the expression levels of p-Akt (Ser 473), nuclear factor kappa B (NF-κB), p-NF-κB (Ser 468), mammalian target of rapamycin (mTOR) and p-mTOR (Ser 2448) protein significantly increased. It is concluded that eEF1A1 may have a carcinogenic effect in T-ALL cells. eEF1A1 expression has noticeable effects on the proliferation enhancement and apoptosis inhibition of Jurkat cells, which may be mediated by the up-regulation of PI3K/Akt/NF-κB and PI3K/Akt/ mTOR signaling pathway.

Analysis of differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia and cervical squamous carcinoma tissues / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and squamous cervical carcinoma (SCC) tissues by differential proteomics, and to provide a basis for studies on CIN molecular pathogenesis, clinical diagnosis and treatment.</p><p><b>METHODS</b>Uterine cervical tissue specimens from the patients treated between August 2008 and September 2009 in the Department of Oncology of Beijing Obstetrics and Gynecology Hospital were collected. There were samples of normal cervix (n = 9), CIN (n = 23, CIN I = 7, CIN II = 8, CIN III = 8) and SCC (n = 7). 2-D DIGE and DeCyder software were used to detect the differentially expressed protein-spots. Then MALDI-TOF/TOF MS was used to analyze the differentially expressed proteins. Collect normal cervix(n = 20), CIN (n = 60) and SCC (n = 20), immunohistochemistry (IHC) and Western blot were used to verify the differentially expressed proteins of S100A9 (S100 calcium-binding protein A9) , eEF1A1 (eukaryotic elongation factor 1-alpha-1) and PKM2 (pyruvate kinase isozymes M2) among the normal cervix, CIN and SCC tissues. Immunohistochemistry was used to detect the differentially expressed S100A9, eEF1A1 and PKM2 in the cervical tissues.</p><p><b>RESULTS</b>2D gel electrophoresis images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 were up-regulated and 19 were down-regulated) were selected among the normal, CIN, and SCC, and 26 proteins were successfully identified. Immunohistochemistry showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0% in normal cervical mucosa, 70.0% in CIN, and 100.0% in squamous cell carcinoma, with a significant difference between them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma. Its positive expression rate was 70.0% in normal cervix, 73.3%in CIN and 60.0% in SCC tissues, with a non-significant difference between them (P = 0.758). The protein PKM2 was mainly expressed in the cell nuclei. Its positive expression rate was 100.0% in normal cervix, 93.3% in CIN and 75.0% in SCC tissues, showing a difference close to statistical significance (P = 0.059) between them. The results of Western blot were similar with that of immunohistochemical examination.</p><p><b>CONCLUSIONS</b>There are differentially expressed proteins among normal cervix, CIN and SCC. S100A9, eEF1A1 and PKM2 may become candidate markers for early diagnosis of cervical cancer and new targets for therapy. It also provides a further basis for studies of the pathogenetic mechanism of CIN developing to cervical cancer.</p>

Effect of knocking down eEF1A1 gene on proliferation and apoptosis in Jurkat cells and its mechanisms / 中国实验血液学杂志

This study was purposed to investigate the effect of knocking down eukaryotic elongation factor 1A1 (eEF1A1) gene on the proliferation and apoptosis in human acute T lymphocytic leukemia (T-ALL) cell line Jurkat and explore its mechanism. The eEF1A1 mRNA and protein expressions of Jurkat cells and 3 healthy adult peripheral blood mononuclear cells (PBMNC) were detected by real time PCR and Western blot, respectively. eEF1A1-shRNA lentivirus was constructed through molecular biological method, and was used to transfect Jurkat cells. Then, cell eEF1A1 mRNA and protein expressions were detected by real time PCR and Western blot, respectively. Cell proliferation, apoptosis and cycle were detected by MTT method, Annexin V-APC labeling and DNA ploidy analysis, respectively. Cell-related protein expressions of phosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway were detected by Western blot. The results showed that eEF1A1 mRNA and protein expression levels of Jurkat cells were significantly higher than that of healthy adult PBMNC, respectively (P < 0.01, P < 0.05). eEF1A1 mRNA and protein expressions of Jurkat cells were significantly knocked down by constructing eEF1A1-shRNA lentivirus. Compared to negative control group (transfected with negative control-shRNA lentivirus), cell proliferation in eEF1A1-shRNA group was significantly inhibited, cell apoptosis was remarkably induced, cell cycle was blocked in G(0)/G(1) phase, and the expression levels of p-Akt (Ser 473), nuclear factor kappa B (NF-κB), p-NF-κB (Ser 468), mammalian target of rapamycin (mTOR) and p-mTOR (Ser 2448) proteins were significantly reduced. It is concluded that eEF1A1 may be a putative oncoprotein in T-ALL cells. Knocking down eEF1A1 gene has noticeable effects on the proliferation inhibition and apoptosis induction of Jurkat cells, which may be mediated by the down-regulation of PI3K/Akt/NF-κB and PI3K/Akt/mTOR signaling pathway.

Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.

Alternative expression and sequence of human elongation factor-1 delta during malignant transformation of human bronchial epithelial cells induced by cadmium chloride / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the alternative expression and sequence of human elongation factor-1 delta (human EF-1 delta p31) during malignant transformation of human bronchial epithelial cells induced by cadmium chloride (CdC12) and its possible mechanism.</p><p><b>METHODS</b>Total RNA was isolated at different stages of transformed human bronchial epithelial cells (16HBE) induced by CdCl2 at a concentration of 5.0 microM. Special primers and probe for human EF-1 delta p31 were designed and expression of human EF-1 delta mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis.</p><p><b>RESULTS</b>The expressions of human EF-1 beta p31 at different stages of 16HBE cells transformed by CdCl2 was elevated (P < 0.01 or P < 0.05). Compared with their corresponding non-transformed cells, the overexpression level of EF-1 delta p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed cells and 7.2 folds in Cd-tumorigenic cells. No change was found n the sequence of overexpressed EF-1beta p31 at different stages of 16HBE cells transformed by CdCl2.</p><p><b>CONCLUSION</b>Overexpression of human EF-1beta p31 is positively correlated with malignant transformation of 16HBE cells induced by CdC12, but is not correlated with DNA mutations.</p>

Identification and Characterization of Gliocladium viride Isolated from Mushroom Fly Infested Oak Log Beds Used for Shiitake Cultivation

A green mold species that has not previously been reported in Korea was isolated from oak log beds used for shiitake (Lentinula edodes) cultivation that were infested by mushroom flies. In this study, we identify the mold species as Gliocladium viride (an anamorph of Hypocrea lutea) and describe its mycological properties. The fungus was cottony on both potato dextrose agar (PDA) and Czapek yeast extract agar (CYA), but was colored white on PDA and became yellowish green and brown on CYA. Mycelial growth on PDA attained a diameter of 73 mm at 30degrees C after 5 days. The fungus grew faster on malt extract agar (> 80 mm, 5 days at 25degrees C) compared to CYA and PDA (< 68 mm, 5 days at 25degrees C). Penicillate conidiophores of the fungus are hyaline, smooth walled, branching above typically in four stages, and 120~240 microm in length. Club-shaped or slender phialides are formed on the metulae. Conidia of the fungus were ovate and elliptic, yellowish brown and green, and 2.5~3.0 microm x 1.8~2.3 microm in size. Typically, slimy conidia are formed in a mass and colored brown to dark green to almost black. The internal transcribed spacer rDNA and translation elongation factor 1 alpha gene sequences of the fungus isolated here show 99% identity with previously identified G. viride strains.

Nuclear elongation factor-I alpha gene a molecular marker for Iranian sandfly identification

Elongation factor-1 alpha, a conserved nuclear protein coding gene was used to identify Iranian sandfly species. The phlebotomine sand flies are the vectors of the parasitic protozoan Leishmania, the causative agents of leishmaniasis, in Iran. Seven sets of primers were tried. PCR amplification of elongation factor-l alpha was successfully achieved for all 14 species of Iranian sandflies that we caught, but different primers had to be used. The aligned DNA sequences of 454 bp [without primers] of the gene had the most similarity to a coding region of the elongation factor-l alpha genes of D. melanogaster, as identified by a BLAST search of GenBank. Each Iranian species, except Phlebotomus caucasicus and P. mongolensis, had a unique combination of nucleotides, i.e. each had a diagnostic sequence. There were no diagnostic sequences for different geographical populations of the species in Iran. We found only a single copy of Ef-l alpha gene in most individual sandflies. However EF-l alpha gene was successfully amplified by PCR but, unfortunately, phylogenetic analysis showed that it might be multicopy in sandflies and so the markers could not be trusted. More highly polymorphic nuclear loci, like microsatellites, might be needed to distinguish morphologically indistinguishable females of the subgenus Paraphlebotomus, e.g. P. caucasiscus from P. mongolensis, in order to resolve their roles as vectors of Leishmania species in gerbils

Comparison of Gene Expression Profile in Eutopic Endometria with or without Endometriosis: A Microarray Study / 대한불임학회지

OBJECTIVE: Pathogenesis of the endometriosis is very complex and the etiology is still unclear. Our hypothesis is that there may be some difference in gene expression patterns between eutopic endometriums with or without endometriosis. In this study, we analyzed the difference of gene expression profile with cDNA microarray. METHODS: Endometrial tissues were gathered from patients with endometriosis or other benign gynecologic diseases. cDNA microarray technique was applied to screen the different gene expression profiles from early and late secretory phase endometria of those two groups. Each three mRNA samples isolated from early and late secretory phase of endometrial tissues of control were pooled and used as master controls and labeled with Cy3-dUTP. Then the differences of gene expression pattern were screened by comparing eutopic endometria with endometriosis, which were labeled with Cy5-dUTP. Fluorescent labeled probes were hybridized on a microarray of 4,800 human genes. RESULTS: Twelve genes were consistently overexpressed in the endometrium of endometriosis such as ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ transporting (ATP5C1) and TNF alpha factor. Eleven genes were consistently down-regulated in the endometriosis samples. Many extracellular matrix protein genes (decorin, lumican, EGF-containing fibulin-like extracellular matrix protein 1, fibulin 5, and matrix Gla protein) and protease/protease inhibitors (serine proteinase inhibitor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1), and insulin like growth factor II associated protein were included. Expression patterns of selected eight genes from the cDNA microarray were confirmed by quantitative RT-PCR or real time RT-PCR. CONCLUSION: The result of this analysis supports the hypothesis that the endometrium from patients with endometriosis has distinct gene expression profile from control endometrium without endometriosis.

Abnormal expression of eukaryotic translation factors in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1delta (TEF-1delta) in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide (NiS).</p><p><b>METHODS</b>Abnormal expressions of human TIF3 and TEF-1delta genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction (RT-PCR) and fluorescent quantitative polymerase chain reaction (FQ-PCR), respectively.</p><p><b>RESULTS</b>RT-PCR analysis primarily showed that both human TIF3 and TEF-1delta mRNA expressions in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-1delta cDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1delta genes were related to malignant degree of the cells induced by nickel.</p><p><b>CONCLUSIONS</b>These findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1delta genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel.</p>

Comparison of RAPD, AFLP, and EF-1alpha Sequences for the Phylogenetic Analysis of Fusarium oxysporum and Its formae speciales in Korea

Although Fursarium oxysporum causes diseases in economically important plant hosts, identification of F. oxysporum formae speciales has been difficult due to confusing phenotypic classification systems. To resolve these complexity, we evaluated genetic relationship of nine formae speciales of F. oxysporum with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and translation elongation factor-1 alpha (EF-1alpha) gene. In addition, the correlation between mycotoxin content of fusaric acid and isolates based on molecular marker data was evaluated using the modified Mantel's test. According to these result, these fusaric acid-producing strains could not identify clearly, and independent of geographic locations and host specificities. However, in the identification of F. oxysporum formae speciales, especially, AFLP analysis showed a higher discriminatory power than that of a the RAPD and EF-1alpha analyses, all three techniques were able to detect genetic variability among F. oxysporum formae speciales in this study.

Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.