The role of GATA switch in benzene metabolite hydroquinone inhibiting erythroid differentiation in K562 cells.

The phenolic metabolite of benzene, hydroquinone (HQ), has potential risks for hematological disorders and hematotoxicity in humans. Previous studies have revealed that reactive oxygen species, DNA methylation, and histone acetylation participate in benzene metabolites inhibiting erythroid differentiation in hemin-induced K562 cells. GATA1 and GATA2 are crucial erythroid-specific transcription factors that exhibit dynamic expression patterns during erythroid differentiation. We investigated the role of GATA factors in HQ-inhibited erythroid differentiation in K562 cells. When K562 cells were induced with 40 µM hemin for 0-120 h, the mRNA and protein levels of GATA1 and GATA2 changed dynamically. After exposure to 40 µM HQ for 72 h, K562 cells were induced with 40 µM hemin for 48 h. HQ considerably reduced the percentage of hemin-induced Hb-positive cells, decreased the GATA1 mRNA, protein, and occupancy levels at α-globin and ß-globin gene clusters, and increased the GATA2 mRNA and protein levels significantly. ChIP-seq analysis revealed that HQ reduced GATA1 occupancy, and increased GATA2 occupancy at most gene loci in hemin-induced K562 cells. And GATA1 and GATA2 might play essential roles in the erythroid differentiation protein interaction network. These results elucidate that HQ decreases GATA1 occupancy and increases GATA2 occupancy at the erythroid gene loci, thereby downregulating GATA1 and upregulating GATA2 expression, which in turn modulates the expression of erythroid genes and inhibits erythroid differentiation. This partially explains the mechanism of benzene hematotoxicity.

Benzene metabolite 1,2,4-benzenetriol changes DNA methylation and histone acetylation of erythroid-specific genes in K562 cells.

1,2,4-Benzenetriol (BT) is one of the phenolic metabolites of benzene, a general occupational hazard and ubiquitous environmental air pollutant with leukemogenic potential in humans. Previous studies have revealed that the benzene metabolites phenol and hydroquinone can inhibit hemin-induced erythroid differentiation in K562 cells. We investigated the roles of DNA methylation and histone acetylation in BT-inhibited erythroid differentiation in K562 cells. When K562 cells were treated with 0, 5, 10, 15 or 20 µM BT for 72 h, hemin-induced hemoglobin synthesis decreased in a concentration-dependent manner. Both 5-aza-2'-deoxycytidine (5-aza-CdR, DNA methyltransferase inhibitor) and trichostatin A (TSA, histone deacetylases inhibitor) could prevent 20 µM BT from inhibiting hemin-induced hemoglobin synthesis and the mRNA expression of erythroid genes. Exposure to BT changed DNA methylation levels at several CpG sites of erythroid-specific genes, as well as the acetylation of histone H3 and H4, chromatin occupancy of GATA-1 and recruitment of RNA polymerase II at α-globin and ß-globin gene clusters after hemin induction. These results demonstrated that BT could inhibit hemin-induced erythroid differentiation, where DNA methylation and histone acetylation also played important roles by down-regulating erythroid-specific genes. This partly explained the mechanisms of benzene hematotoxicity.

[Laboratory design and practice for undergraduates: Using RNAi to modulate gene expression].

RNA interference is a gene silencing phenomenon mediated by short double-stranded RNAs, which has become a widely used research technology for reverse genetics. In order to make students understand the technology better, the students were required to select target genes, to design small interfering RNAs (siRNAs) and primers, and then to test the effect of gene silencing mediated by siRNAs. Taking the fifth group in 2018 as an example, Mus musculus acyl-CoA synthetase long-chain family member 1 (Acsl1) was selected as the target gene, two pairs of siRNAs targeting Acsl1 mRNA were designed and transfected into 3T3-L1 by electroporation, then the total RNAs were extracted and synthesized to cDNA, and the expression levels of mRNAs were finally tested by relative quantitative PCR. The results showed that both pairs of siRNAs had more than 60% silencing effects. In the past three years, about 83% of the students completed all the experiments successfully and screened out at least a pair of effective siRNA. This teaching practice for undergraduates enhances students' understanding of RNA interference principle and technology, and exercises students' lab experience and scientific research ability.

Inhibition of Erythroid Differentiation of Human Leukemia K562 Cells by N-acetylcysteine and Ascorbic Acid through Downregulation of ROS.

This study investigated the effects of N-acetylcysteine (NAC) and ascorbic acid (AA) on hemin-induced K562 cell erythroid differentiation and the role of reactive oxygen species (ROS) in this process. Hemin increased ROS levels in a concentration-dependent manner, whereas NAC and AA had opposite effects. Both NAC and AA eliminated transient increased ROS levels after hemin treatment, inhibited hemin-induced hemoglobin synthesis, and decreased mRNA expression levels of ß-globin, γ-globin, and GATA-1 genes significantly. Pretreatment with 5,000 µmol/L AA for 2 h resulted in a considerably lower inhibition ratio of hemoglobin synthesis than that when pretreated for 24 h, whereas the ROS levels were the lowest when treated with 5,000 µmol/L AA for 2 h. These results show that NAC and AA might inhibit hemin-induced K562 cell erythroid differentiation by downregulating ROS levels.

High glucose induced endothelial to mesenchymal transition in human umbilical vein endothelial cell.

BACKGROUND: Studies have shown that endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis. The aim of this study was to investigate the influence of high glucose and related mechanism of MAPK inhibitor or specific antioxidant on the EndMT. METHODS: In vitro human umbilical vein endothelial cells (HUVEC) were cultured with 11mM, 30mM, 60mM and 120mM glucose for 0, 24, 48, 72 and 168h. Endothelial cell morphology was observed with microscope, and RT-PCR was used to detect mRNA expression of endothelial markers VE-cadherin and CD31, mesenchymal markers α-SMA and collagen I, and transforming growth factor TGF-ß1. Immunofluorescence staining was performed to detect the expression of CD31 and α-SMA. The concentration of TGF-ß1 in the supernatant was detected by ELISA. ERK1/2 phosphorylation level was detected by Western blot analysis. RESULTS: High glucose induced EndMT and increased the TGF-ß1 level in HUVEC cells. Cells in high glucose for 7 days showed a significant decrease in mRNA expression of CD31 and VE-cadherin, and a significant increase in that of α-SMA and collagen I, while lost CD31 staining and acquired α-SMA staining. ERK signaling pathway blocker PD98059 significantly attenuated the high glucose-induced increase in the ERK1/2 phosphorylation level. PD98059 and NAC both inhibited high glucose-induced TGF-ß1 expression and attenuated EndMT marker protein synthesis. CONCLUSION: High glucose could induce HUVEC cells to undergo EndMT. NAC and ERK signaling pathway may play important role in the regulation of the TGF-ß1 biosynthesis during high glucose-induced EndMT.

RTL-P: a sensitive approach for detecting sites of 2'-O-methylation in RNA molecules.

2'-O-methylation is present within various cellular RNAs and is essential to RNA biogenesis and functionality. Several methods have been developed for the identification and localization of 2'-O-methylated sites in RNAs; however, the detection of RNA modifications, especially in low-abundance RNAs and small non-coding RNAs with a 2'-O-methylation at the 3'-end, remains a difficult task. Here, we introduce a new method to detect 2'-O-methylated sites in diverse RNA species, referred to as RTL-P [Reverse Transcription at Low deoxy-ribonucleoside triphosphate (dNTP) concentrations followed by polymerase chain reaction (PCR)] that demonstrates precise mapping and superior sensitivity compared with previous techniques. The main procedures of RTL-P include a site-specific primer extension by reverse transcriptase at a low dNTP concentration and a semi-quantitative PCR amplification step. No radiolabeled or fluorescent primers are required. By designing specific RT primers, we used RTL-P to detect both previously identified and novel 2'-O-methylated sites in human and yeast ribosomal RNAs (rRNAs), as well as mouse piwi-interacting RNAs (piRNAs). These results demonstrate the powerful application of RTL-P for the systematic analysis of fully or partially methylated residues in diverse RNA species, including low-abundance RNAs or small non-coding RNAs such as piRNAs and microRNAs (miRNAs).

Phenolic metabolites of benzene induced caspase-dependent cytotoxicities to K562 cells accompanied with decrease in cell surface sialic acids.

Benzene-induced erythropoietic depression has been proposed to be due to the production of toxic metabolites. Presently, the cytotoxicities of benzene metabolites, including phenol, catechol, hydroquinone, and 1,2,4-benzenetriol, to erythroid progenitor-like K562 cells were investigated. After exposure to these metabolites, K562 cells showed significant inhibition of viability and apoptotic characteristics. Each metabolite caused a significant increase in activities of caspase-3, -8, and -9, and pretreatment with caspase-3, -8, and -9 inhibitors significantly inhibited benzene metabolites-induced phosphatidylserine exposure. These metabolites also elevated expression of Fas and FasL on the cell surface. After exposure to benzene metabolites, K562 cells showed an increase in reactive oxygen species level, and pretreatment with N-acetyl-l-cysteine significantly protected against the cytotoxicity of each metabolite. Interestingly, the control K562 cells and the phenol-exposed cells aggregated together, but the cells exposed to other metabolites were scattered. Further analysis showed that hydroquione, catechol, and 1,2,4-benzenetriol induced a decrease in the cell surface sialic acid levels and an increase in the cell surface sialidase activity, but phenol did not cause any changes in sialic acid levels and sialidase activity. Consistently, an increase in expression level of sialidase Neu3 mRNA and a decrease in mRNA level of sialyltransferase ST3GAL3 gene were detected in hydroquione-, catechol-, or 1,2,4-benzenetriol-treated cells, but no change in mRNA levels of two genes were found in phenol-treated cells. In conclusion, these benzene metabolites could induce apoptosis of K562 cells mainly through caspase-8-dependent pathway and ROS production, and sialic acid metabolism might play a role in the apoptotic process.

The role of ROS in hydroquinone-induced inhibition of K562 cell erythroid differentiation.

The role of ROS in hydroquinone-induced inhibition of K562 cell erythroid differentiation was investigated. After K562 cells were treated with hydroquinone for 24 h, and hemin was later added to induce erythroid differentiation for 48 h, hydroquinone inhibited hemin-induced hemoglobin synthesis and mRNA expression of γ-globin in K562 cells in a concentration-dependent manner. The 24-h exposure to hydroquinone also caused a concentration-dependent increase at an intracellular ROS level, while the presence of N- acetyl-L-cysteine prevented hydroquinone- induced ROS production in K562 cells. The presence of N-acetyl-L-cysteine also prevented hydroquinone inhibiting hemin-induced hemoglobin synthesis and mRNA expression of γ-globin in K562 cells. These evidences indicated that ROS production played a role in hydroquinone-induced inhibition of erythroid differentiation.

Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells.

Objective: Hydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. Methods: In this study, we treated K562 cells with 40 µmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. Results: One hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. Conclusion: Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation.

Role of microRNA-regulated cancer stem cells in recurrent hepatocellular carcinoma.

Among the most common cancers, hepatocellular carcinoma (HCC) has a high rate of tumor recurrence, tumor dormancy, and drug resistance after initial successful chemotherapy or radiotherapy. A small subset of cancer cells, cancer stem cells (CSCs), exhibit stem cell characteristics and are present in various cancers, including HCC. The dysregulation of microRNAs (miRNAs) often accompanies the occurrence and development of HCC. miRNAs can influence tumorigenesis, progression, recurrence, and drug resistance by regulating CSCs properties, which supports their clinical utility in managing and treating HCC. This review summarizes the regulatory effects of miRNAs on CSCs in HCC with a special focus on their impact on HCC recurrence.

Identification of potential pathways and microRNA-mRNA networks associated with benzene metabolite hydroquinone-induced hematotoxicity in human leukemia K562 cells.

BACKGROUND: Hydroquinone (HQ) is a phenolic metabolite of benzene with a potential risk for hematological disorders and hematotoxicity in humans. In the present study, an integrative analysis of microRNA (miRNA) and mRNA expressions was performed to identify potential pathways and miRNA-mRNA network associated with benzene metabolite hydroquinone-induced hematotoxicity. METHODS: K562 cells were treated with 40 µM HQ for 72 h, mRNA and miRNA expression changes were examined using transcriptomic profiles and miRNA microarray, and then bioinformatics analysis was performed. RESULTS: Out of all the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) induced by HQ, 1482 DEGs and 10 DEMs were up-regulated, and 1594 DEGs and 42 DEMs were down-regulated. HQ-induced DEGs were involved in oxidative stress, apoptosis, DNA methylation, histone acetylation and cellular response to leukemia inhibitory factor GO terms, as well as metabolic, Wnt/ß-catenin, NF-κB, and leukemia-related pathways. The regulatory network of mRNAs and miRNAs includes 23 miRNAs, 1108 target genes, and 2304 potential miRNAs-mRNAs pairs. MiR-1246 and miR-224 had the potential to be major regulators in HQ-exposed K562 cells based on the miRNAs-mRNAs network. CONCLUSIONS: This study reinforces the use of in vitro model of HQ exposure and bioinformatic approaches to advance our knowledge on molecular mechanisms of benzene hematotoxicity at the RNA level.

Mediating effect analysis of visceral adiposity index on free triiodothyronine to free thyroxine ratio and non-alcoholic fatty liver disease in euthyroid population.

Background: The association between free triiodothyronine/free thyroxine (FT3/FT4) and non-alcoholic fatty liver disease (NAFLD) in euthyroid subjects is unclear. In addition, few studies have explored whether VAI mediates the association between FT3/FT4 ratio and NAFLD in the euthyroid population. We aimed to analyze the mediating effect of VAI on the FT3/FT4 ratio and NAFLD risk in the euthyroid population. Methods: This cross-sectional study included 7 946 annual health examinees from the Health Examination Center, Hebei General Hospital, from January to December 2020. The basic information and biochemical parameters, as well as calculated FT3/FT4 ratio and VAI were collected. NAFLD was diagnosed according to abdominal ultrasonography. The fibrosis score for NAFLD positive subjects (NFS) was calculated to reflect the extent of liver fibrosis. The risk of NAFLD was analyzed by quartiles of FT3/FT4 ratio (Q1-Q4 quartiles) and VAI (V1-V4 quartiles), respectively. Pearson correlation analysis was performed to investigate the correlation between FT3/FT4 ratio and VAI. Multivariate logistic regression analysis was applied to analyze the effect of FT3/FT4 ratio and VAI on NAFLD and NFS status. Bootstrap was conducted to explore whether VAI mediated the association between FT3/FT4 ratio and NAFLD. Results: Of the 7 946 participants, 2 810 (35.36%) had NAFLD and 5 136 (64.64%) did not. Pearson correlation analysis indicated that FT3/FT4 ratio was positively associated with VAI (P<0.05). Multivariate logistic regression analysis indicated that compared to the Q1 group, the risk of NAFLD significantly increased in Q3 group [OR=1.255, 95%CI (1.011, 1.559)] and Q4 group [OR=1.553, 95%CI (1.252, 1.926)](P<0.05). Compared to the V1 group, the risk of NAFLD notably increased in V2 group [OR=1.584, 95%CI (1.205, 2.083)], V3 group [OR=2.386, 95%CI (1.778, 3.202)] and V4 group [OR=4.104, 95%CI (2.835, 5.939)] (P<0.01). There was no relevance between FT3/FT4 ratio, VAI and NFS status. Mediating effect analysis showed that FT3/FT4 ratio significantly directly influenced NAFLD prevalence [ß=3.7029, 95%CI (2.9583, 4.4474)], and VAI partly mediated the indirect effect of the FT3/FT4 ratio on NAFLD prevalence [ß=2.7649, 95%CI (2.2347, 3.3466)], and the mediating effect accounted for 42.75% of the total effects. Conclusion: Both FT3/FT4 ratio and VAI were predictors of NAFLD, and VAI partly mediated the indirect effect of the FT3/FT4 ratio on NAFLD prevalence in the euthyroid population.

SnoRNAs from the filamentous fungus Neurospora crassa: structural, functional and evolutionary insights.

BACKGROUND: SnoRNAs represent an excellent model for studying the structural and functional evolution of small non-coding RNAs involved in the post-transcriptional modification machinery for rRNAs and snRNAs in eukaryotic cells. Identification of snoRNAs from Neurospora crassa, an important model organism playing key roles in the development of modern genetics, biochemistry and molecular biology will provide insights into the evolution of snoRNA genes in the fungus kingdom. RESULTS: Fifty five box C/D snoRNAs were identified and predicted to guide 71 2'-O-methylated sites including four sites on snRNAs and three sites on tRNAs. Additionally, twenty box H/ACA snoRNAs, which potentially guide 17 pseudouridylations on rRNAs, were also identified. Although not exhaustive, the study provides the first comprehensive list of two major families of snoRNAs from the filamentous fungus N. crassa. The independently transcribed strategy dominates in the expression of box H/ACA snoRNA genes, whereas most of the box C/D snoRNA genes are intron-encoded. This shows that different genomic organizations and expression modes have been adopted by the two major classes of snoRNA genes in N. crassa . Remarkably, five gene clusters represent an outstanding organization of box C/D snoRNA genes, which are well conserved among yeasts and multicellular fungi, implying their functional importance for the fungus cells. Interestingly, alternative splicing events were found in the expression of two polycistronic snoRNA gene hosts that resemble the UHG-like genes in mammals. Phylogenetic analysis further revealed that the extensive separation and recombination of two functional elements of snoRNA genes has occurred during fungus evolution. CONCLUSION: This is the first genome-wide analysis of the filamentous fungus N. crassa snoRNAs that aids in understanding the differences between unicellular fungi and multicellular fungi. As compared with two yeasts, a more complex pattern of methylation guided by box C/D snoRNAs in multicellular fungus than in unicellular yeasts was revealed, indicating the high diversity of post-transcriptional modification guided by snoRNAs in the fungus kingdom.

Analysis of the clinical characteristics and refraction state in premature infants: a 10-year retrospective analysis.

AIM: To report the visual outcomes and refractive status in premature infants with and without retinopathy of prematurity (ROP) who were or not treated. METHODS: The clinical records of all premature infants with or without ROP and with or without treatment between 2007 and 2017 were retrospectively reviewed. Basic demographic data, serial changes in ROP incidence, treatment and outcomes, and the refractive states were analyzed. Correlations among myopia and astigmatism progression, birth weight, gestational age, and treatment methods were also analyzed. RESULTS: A total of 562 screened premature infants (all Chinese, 1124 eyes), were recruited with a 378:184 male-to-female ratio. Birth weight did not directly influence ROP incidence. The overall ROP incidence was 16.55% (93/562 cases). The incidences in boys and girls were 16.14% (33/378 cases) and 17.39% (32/184 cases), respectively, and this difference was not significant. However, all infants with serious ROP (stage IV and V) were male. Myopia combined with astigmatism was common in premature infants with and without ROP (30.99%, 172/555 cases), and myopic refraction (including myopia and myopia combined with astigmatism) was more common in premature infants with ROP (48.84%, 42/86 cases). In the >8.00 diopter group, there were significantly more ROP infants than without ROP. Myopic refraction (including myopia and myopia combined with astigmatism) was most common in infants with ROP after treatment (63.63%, 7/11 cases). CONCLUSION: The refractive state is different between premature infants and mature infants. Those treated for ROP had a higher chance of developing myopia, astigmatism, and higher diopter.

The rat mitochondrial Ori L encodes a novel small RNA resembling an ancestral tRNA.

The RNA minihelix, a proposed tRNA precursor, exhibits tRNA-like properties. Sequence-specific RNA minihelices can inhibit cell growth probably due to their binding to the cognate tRNA and naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose. Thus far, no natural RNA minihelices have been found. In the present study, we found a novel small RNA of 32 nucleotides, which is expressed abundantly in all rat tissues tested. Distinct from all of known endogenous small RNAs, this small RNA (temporarily named as tpl-sRNA) can form an RNA minihelix containing a stem-loop domain followed by ACCA. tpl-sRNA is encoded by the light-strand replication origin (Ori L) of the rat mitochondrial genome, and the 3'-terminal CCA of tpl-sRNA is post-transcriptionally added. Moreover, tpl-sRNA is chargeable in vivo. Our study demonstrates for the first time an endogenous small RNA that resembles an ancestral tRNA and exhibits some tRNA-like properties in mammals.

A novel experimental approach for systematic identification of box H/ACA snoRNAs from eukaryotes.

Box H/ACA snoRNAs represent an abundant group of small non-coding RNAs mainly involved in the pseudouridylation of rRNAs and/or snRNAs in eukaryotes and Archaea. In this study, we describe a novel experimental method for systematic identification of box H/ACA snoRNAs from eukaryotes. In the specialized cDNA libraries constructed by this method with total cellular RNAs from human blood cells, the high efficiency of cloning for diverse box H/ACA snoRNAs was achieved and seven novel species of this snoRNA family were identified from human for the first time. Furthermore, the novel method has been successfully applied for the identification of the box H/ACA snoRNAs from Drosophila and the fission yeast, demonstrating a powerful ability for systematic analysis of box H/ACA snoRNAs in a broad spectrum of eukaryotes.

Changes in DNA methylation of erythroid-specific genes in K562 cells exposed to catechol in long term.

Catechol is one of phenolic metabolites of benzene that is a general occupational hazard and a ubiquitous environmental air pollutant. Catechol also occurs naturally in fruits, vegetables and cigarettes. Previous studies have revealed that 72h exposure to catechol improved hemin-induced erythroid differentiation of K562 cells accompanied with elevated methylation in erythroid specific genes. In present study, K562 cells were treated with 0, 10 or 20µM catechol for 1-4weeks, hemin-induced hemoglobin synthesis increased in a concentration- and time-dependent manner and the enhanced hemoglobin synthesis was relatively stable. The mRNA expression of α-, ß- and γ-globin genes, erythroid heme synthesis enzymes PBGD and ALAS2, transcription factor GATA-1 and NF-E2 showed a significant increase in K562 cells exposed to 20µM catechol for 3w, and catechol enhanced hemin-induced mRNA expression of these genes. Quantitative MassARRAY methylation analysis also confirmed that the exposure to catechol changed DNA methylation levels at several CpG sites in several erythroid-specific genes and their far upstream of regulatory elements. These results demonstrated that long-term exposure to low concentration of catechol enhanced the hemin-induced erythroid differentiation of K562 cells, in which DNA methylation played a role by up-regulating erythroid specific genes.

High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by cultivation-independent approach.

Heterosmilax japonica Kunth is well recognized for its diuretic effects in China. However, little is known about its endophytic fungi. In this study, microbial communities inhabiting the stems of H. japonica in spring and summer were investigated by light microscopy and cultivation-independent approaches, such as RFLP analysis and sequencing of rDNA ITS library. Molecular phylogenetic analysis showed that a broad spectrum of fungi, including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia, is able to colonize the plants internally. Particularly, several rDNA sequences determined in this study like YJ4-61 are not specifically affiliated with any currently documented fungal sequences in the public database. Several sequence types, such as YJ4-9 and YJ4-70, are significantly similar to some uncultured environmental samples. Furthermore, our result also showed that the samples collected in spring harbored more abundant endophytic populations than that in summer, implying a seasonal fluctuation for the endophytes in H. japonica.

[Role of exogenous Ca2+ in the somatic embryogenesis of Lycium barbarum L].

In this study, Embryogenic and non-embryogenic calli were separately obtained by cultivation of leaf segments on MS medium containing and not containing 2,4-D 0.2 mg/L. The calli were transferred to an 2,4-D-free MS medium containing different concentrations of (45)Ca(2+) and EGTA cultured, and microscopic examination of tissue sliced, gamma-ray energy spectrum analysis, ELISA and two-dimensional polyacrylamide-gel electrophoresis were used to study the relation between changes in Ca(2+) concentration and protein composition changes during somatic embryogenesis. The results showed that: (1) Calli of dedifferentiation were obtained by cultivating in the inductive medium (MS+2,4-D 0.2 mg/L) and then transferred to the 2,4-D-free (MS) differentiation medium. After cultivating, the large number of embryogenic cells divided and somatic embryogenic calli (EC) were formed; embryogenic cell differentiation and somatic embryo ware not formed when the dedifferentiation calli, which were cultivated in the inductive medium without 2,4-D, ware transferred to the cultivating of differentiation, so calli were called non-embryogenic calli (NEC). (2) SE frequency of EC was rised with exogenous Ca(2+) concentration was going up, and adding peak value (70.5% to 74.5%) when Ca(2+)concentration was from 0.8 to 1.6 mmol/L, then SE frequency was dropped markedly with Ca(2+) concentration was farther increasing. Formation of meristematic cell aggregates of NEC was also enhanced when exogenous Ca(2+) concentration was from 0.8 to 1.6 mmol/L. (3) After adding EGTA, which was Ca(2+) antagonist, SE frequency was dropped markedly, and SE frequency was fallen along with increased of EGTA concentration. When EGTA concentration went up to 1.2 micromol/L, SE frequency dropped to 5%, and the formation of meristematic cell aggregates on NEC was inhibited. (4) When 2 microCi (45)Ca(2+)/mL was added, the uptake of (45)Ca(2+) by EC and NEC was different, two uptake peaks of (45)Ca(2+)appeared in EC at the embryogenic cell differentiation of stage, and the uptake of (45)Ca(2+) of EC was 4-5 times higher than that of NEC, and the uptake frequency of (45)Ca(2+) improved from 54.1% to 74.5%. The uptake of (45)Ca(2+) by NEC during development not only was lower than that by EC but also there were no such marked peak as those with EC. (5) The CaM content examined by ELISA was increased markedly at multi-cellular embryo and globular embryoid stage of EC. After adding Ca(2+), the CaM content increased significantly, the CaM content of EC was 2-3 times that of NEC. (6) The IEF/SDS-PAGE results showed that the numbers and amount of protein components were widely different between the two kinds of callus with different morphogenesis patterns, the number of proteins of EC had more components than those of NEC. The largest differences protein species presented with Ca(2+) ware added, the more proteins presented on the range of molecular weight was from 43 kD to 66 kD and pI values was from 4.0 to 7.0.