Three novel Actinoplanes species isolated by using polyaspartic acid as a water-retaining agent for the enrichment in situ.

Three novel actinomycete strains, designated TRM66264-DLMT, TRM88002T and TRM88003T, were isolated by using polyaspartic acid as a water-retaining agent for the enrichment in situ. The 16S rRNA gene sequence and phylogenetic analyses of three strains indicated that they belonged to the genus Actinoplanes. The phylogenetically closest strains of TRM66264-DLMT, TRM88002T and TRM88003T were Actinoplanes bogorensis LIPI11-2-Ac043T (98.4 %), Actinoplanes abujensis A4029T (98.0 %) and Actinoplanes ferrugineus IFO15555T (98.1 %), respectively. The major polar lipids of strains TRM66264-DLMT and TRM88002T were phosphatidylethanolamine and disphosphatidylglycerol, while strain TRM88003T only had phosphatidylethanolamine. The predominant menaquinones of strain TRM66264-DLMT were identified as MK-9(H4) and MK-9 (H6). Strains TRM88002T and TRM88003T had MK-9(H4). The cell-wall peptidoglycan of three strains contained meso-diaminopimelic acid. The whole-cell sugars of strain TRM66264-DLMT were identified as arabinose, glucose, galactose and xylose. Strains TRM88002T and TRM88003T mainly had arabinose and glucose. The DNA G+C content of strains TRM66264-DLMT, TRM88002T and TRM88003T were 70.48, 70.46 and 70.64 mol%, respectively. Genotypic and phenotypic analysis confirmed that all three strains sre new members of the genus Acinoplanes. Therefore, it is proposed that strains TRM66264-DLMT, TRM88002T and TRM88003T represent three novel species of the genus Actinoplanes, for which the names Actinoplanes polyasparticus sp. nov. (type strain TRM66264-DLMT=CCTCC AA 2021015T=LMG 32389T), Actinoplanes hotanensis sp. nov. (type strain TRM88002T=CCTCC AA 2021036T=LMG 32621T) and Actinoplanes aksuensis sp. nov. (type strain TRM88003T=CCTCC AA 2021037 T=LMG 32622T) are proposed.

Rhizobium alarense sp. nov. and Rhizobium halophilum sp. nov. isolated from the nodule and rhizosphere of Lotus japonicus.

Two strains (TRM95111T and TRM95001T) of Gram-stain-negative, aerobic, rod-shaped microbes were isolated from the nodule and rhizosphere of Lotus japonicus grown in the campus of Tarim University in Alar, Xinjiang, China. Strain TRM95111T and strain TRM95001T shared 93.1% 16S rRNA gene sequences similarity with each other and had 98.2 and 97.9% 16S rRNA gene sequence similarity to the closest species Rhizobium subbaraois JC85T and R. halotolerans AB21T by EzBioCloud blast, respectively. Phylogenetic analyses based on 16S rRNA gene sequences, housekeeping gene sequences and core-proteome average amino acid identity (cpAAI) showed that two strains belonged to the genus Rhizobium. The value of digital DNA-DNA hybridization (dDDH) between strain TRM95111T and the closest strain R. subbaraonis JC85T was 21.8%, respectively. The dDDH value between strain TRM95001T and the closest strains R. tarimense PL-41T was 27.1%. Whole-genome average nucleotide identity (ANI) values of the strain TRM95111T were 75.6-79.3% and strain TRM95001T were 79.2-83.8%, compared to their closely related strains. The G + C content values of strain TRM95111T and TRM95001T were 65.1 and 60.7 mol%, respectively. Two isolates contained predominant quinone of Q-10 and the major fatty acids was C18:1ω7c and they were sensitive to 1 µg of amikacin and kanamycin. The polar lipids of strain TRM95111T included unidentified aminophospho lipids (APL1-3), unidentified phospholipids (PL1-2), phosphatidylcholine (PC), unidentified lipids (L1-5) and phospholipids of unknown structure containing glucosamine (NPG), compared to the polar lipids of strain TRM95001T including unidentified aminophospho lipids (APL1-3), unidentified phospholipids (PL1-2), phosphatidylcholine (PC), unidentified lipids (L2-5), hydroxy phosphatidyl ethanolamine (OH-PE) and phospholipids of unknown structure containing glucosamine (NPG). Nodulation tests showed that two strains could induce nodules formation in L. japonicus. Based on the genomic, phenotypic and phylogenetic analyses, strain TRM95111T and strain TRM95001T are suggested to represent two new species of the genus Rhizobium, whose names are proposed as Rhizobium alarense sp. nov. and Rhizobium halophilum sp. nov. The type strains are TRM95111T (=CCTCC AB 2021116T =JCM34826T) and TRM950011T (=CCTCC AB 2021095T =JCM34967T), respectively.

An aquaporin PvTIP4;1 from Pteris vittata may mediate arsenite uptake.

The fern Pteris vittata is an arsenic hyperaccumulator. The genes involved in arsenite (As(III)) transport are not yet clear. Here, we describe the isolation and characterization of a new P. vittata aquaporin gene, PvTIP4;1, which may mediate As(III) uptake. PvTIP4;1 was identified from yeast functional complement cDNA library of P. vittata. Arsenic toxicity and accumulating activities of PvTIP4;1 were analyzed in Saccharomyces cerevisiae and Arabidopsis. Subcellular localization of PvTIP4;1-GFP fusion protein in P. vittata protoplast and callus was conducted. The tissue expression of PvTIP4;1 was investigated by quantitative real-time PCR. Site-directed mutagenesis of the PvTIP4;1 aromatic/arginine (Ar/R) domain was studied. Heterologous expression in yeast demonstrates that PvTIP4;1 was able to facilitate As(III) diffusion. Transgenic Arabidopsis showed that PvTIP4;1 increases arsenic accumulation and induces arsenic sensitivity. Images and FM4-64 staining suggest that PvTIP4;1 localizes to the plasma membrane in P. vittata cells. A tissue location study shows that PvTIP4;1 transcripts are mainly expressed in roots. Site-directed mutation in yeast further proved that the cysteine at the LE1 position of PvTIP4;1 Ar/R domain is a functional site. PvTIP4;1 is a new represented tonoplast intrinsic protein (TIP) aquaporin from P. vittata and the function and location results imply that PvTIP4;1 may be involved in As(III) uptake.

Engineering arsenic tolerance and hyperaccumulation in plants for phytoremediation by a PvACR3 transgenic approach.

Arsenic (As) pollution is a global problem, and the plant-based cleanup of contaminated soils, called phytoremediation, is therefore of great interest. Recently, transgenic approaches have been designed to develop As phytoremediation technologies. Here, we used a one-gene transgenic approach for As tolerance and accumulation in Arabidopsis thaliana . PvACR3, a key arsenite [As(III)] antiporter in the As hyperaccumulator fern Pteris vittata , was expressed in Arabidopsis , driven by the CaMV 35S promoter. In response to As treatment, PvACR3 transgenic plants showed greatly enhanced tolerance. PvACR3 transgenic seeds could even germinate and grow in the presence of 80 µM As(III) or 1200 µM arsenate [As(V)] treatments that were lethal to wild-type seeds. PvACR3 localizes to the plasma membrane in Arabidopsis and increases arsenite efflux into external medium in short-term experiments. Arsenic determination showed that PvACR3 substantially reduced As concentrations in roots and simultaneously increased shoot As under 150 µM As(V). When cultivated in As(V)-containing soil (10 ppm As), transgenic plants accumulated approximately 7.5-fold more As in above-ground tissues than wild-type plants. This study provides important insights into the behavior of PvACR3 and the physiology of As metabolism in plants. Our work also provides a simple and practical PvACR3 transgenic approach for engineering As-tolerant and -hyperaccumulating plants for phytoremediation.

Micromonospora profundi TRM 95458 converts glycerol to a new osmotic compound.

Plant growth and agricultural productivity was greatly limited by soil salinity and alkalization. The application of salt-tolerant rhizobacteria could effectively improve plant tolerance to saline-alkali stress. Micromonospora profundi TRM 95458 was obtained from the rhizosphere of chickpea (Cicer arietinum L.) as a moderate salt-tolerant rhizobacteria. A new osmotic compound (ABAGG) was isolated from the fermentation broth of M. profundi TRM 95458. The chemical structure of the new compound was elucidated by analyzing nuclear magnetic resonance (NMR) and high-resolution mass (HRMS) data. M. profundi TRM 95458 could convert glycerol into ABAGG. The accumulation of ABAGG varied depending on the amount of glycerol and glycine added to the fermentation medium. In addition, the concentration of NaCl affected the ABAGG content obviously. The highest yield of ABAGG was observed when the salt content of the fermentation medium was 10 g/L. The study indicated that salt stress led to the accumulation of ABAGG using glycerol and glycine as substrates, suggesting ABAGG might aid in the survival and adaptation of the strain in saline-alkaline environments as a new osmotic compound.

Plasma sFlt-1-to-PlGF ratio is correlated with inflammatory but not with oxidative stress in Chinese preeclamptic women.

PURPOSE: Considerable interest has been focused on angiogenic factors and angiogenic imbalance in the field of pre-eclampsia (PE), owing to its gaining role in the development of PE. This study was addressed to investigate the associations of sFlt-1-to-PlGF plasma ratios with oxidative stress assessed by the level of 8-isoprostane, and inflammation measured by the level of high-sensitive C-reactive protein (hs-CRP), and adipocytokines. METHODS: A total of 83 patients with PE including 47 mild PE (MPE) and 36 severe PE (SPE) and 50 age-matched normotensive subjects in the third trimester of pregnancy were examined. Measurements included body mass index (BMI), systolic and diastolic blood pressure (BP) levels, plasma concentrations of hs-CRP, 8-isoprostane, adiponectin, and leptin. RESULTS: Subjects with PE had higher levels of sFlt-1/PlGF (P < 0.01), hs-CRP (P < 0.01), 8-isoprostane and leptin (both P < 0.01) and lower adiponectin (P < 0.01) than did normotensive control subjects. Significant positive correlations were found between plasma sFlt-1/PlGF and hs-CRP (r = 0.437, P < 0.01) or leptin (r = 0.656, P < 0.01). A weak inverse correlation emerged between sFlt-1/PlGF and adiponectin (r = -0.306, P < 0.01). When a multiple regression analysis was performed, with sFlt-1/PlGF as a dependent variable and all the other parameters as independent variables, sFlt-1/PlGF maintain a significant relationship with leptin (beta = 0.219, P < 0.05) and with hs-CRP (beta = 0.295, P < 0.01) as well as with systolic BP(beta = 0.446, P < 0.05). CONCLUSIONS: In Chinese preeclamptic women, plasma sFlt-1-to-PlGF ratio is correlated with inflammatory and adipocytokines but not with oxidative stress.

[Effect of extracellular signal-regulated kinase pathway on nitric oxide release by human umbilical vein endothelial cell induced by placental growth factor-1].

OBJECTIVE: To investigate the effect of extracellular signal-regulated kinase (ERK) pathway on nitric oxide (NO) release by human umbilical vein endothelial cell (HUVEC) induced by placental growth factor-1 (PLGF-1). METHODS: During January to April 2006, 50 samples of umbilical vein blood were collected from newborns delivered by cesarean section due to intrauterine distress and abnormal fetal position. HUVEC were primarily cultured by trypsin digestion. Then the following procedures were performed: (1) Cells were identified using the morphology and VIII factor immunohistochemistry methods if the culture was satisfactory. (2) Cells were collected, and fms-like tyrosin kinase (Flt-1) protein and its mRNA expression were detected with immunoprints and RT-PCR methods. (3) The protein was extracted after cells were treated with PLGF-1 (cells were collected before the treatment and 2.5, 5, 10, 20 min after the treatment). The protein levels of ERK were determined by immunoprints. (4) The cells were cultured with serum-free culture medium containing PLGF-1 only (culture media were collected 20, 40, 160, 360, 480, 720 and 1440 min after the treatment). The quantity of NO was detected with nitrate reductase method. (5) The cells were cultured with serum-free culture medium containing PD98059, the inhibitor of mitogen-activated protein kinase (MAPK)/MEK for 60 min. Then the cells were cultured continuously with the serum-free culture medium containing PLGF-1 for 60 min. The culture media were collected. The quantity of NO was detected by nitrate reductase method. The samples were divided into treatment group and control group. Control group was exactly the same in treatment time, culture condition, and time to collect the cells as the treatment group, except that it was not treated with PLGF-1 or PD 98059. RESULTS: (1) By morphology and VIII factor immunohistochemistry the cultured cells were identified to be HUVEC. (2) Flt-1 mRNA and protein were expressed in HUVEC. (3) Expression of ERK protein started to increase at 2.5 min after treatment of HUVEC with PLGF-1, reaching the peak at 5 min, and decreased at 10 min. (4) In comparison with the control group, NO started to increase at 20 min after treatment of HUVEC with PLGF-1 (6.96 +/- 0.34) micromol/L, significantly increased at 40 min (9.45 +/- 0.59) micromol/L, and arrived at the peak at 480 min (15.82 +/- 0.69) micromol/L Comparison between the two groups showed a significant difference (P<0.05). (5) Release of NO from the cells treated with PD98059 for 1 hour and PLGF was significantly inhibited, compared with the cells treated with PLGF-1 only. CONCLUSION: ERK pathways play an important role in NO release by HUVEC induced by PLGF.

CRISPR as a strong gene editing tool.

Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non- Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human. [BMB Reports 2017; 50(1): 20-24].

Expression of endothelial nitric oxide synthase traffic inducer in the placenta of pregnancy induced hypertension.

The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) in the placenta of the patients with pregnancy induced hypertension (PIH) was detected and its role in the pathogenesis of PIH was studied. The pathological changes in placental vessels were observed by HE staining. NO2-/NO3-, the stable metabolic end products of NO, was measured with nitrate reductase. The eNOS activity in placental tissues was assayed by spectrophotometry. Western blot analysis was applied to detect NOSTRIN expression. The incidence of thickening and fibronoid necrosis of placental vessels was significantly higher in women with PIH than in the normal group (P < 0.01). The levels of placental NO2-/NO3- in PIH patients (27.53 +/- 7.48 micromol/mg) were significantly lower than in normal group (54.27 +/- 9.53 micromol/mg, P < 0.01). The activity of eNOS was significantly decreased in PIH group (12.826 +/- 3.61 U/mg) as compared with that in normal group (21.72 +/- 3.83 U/mg, P < 0.01). Western blot analysis revealed that both groups expressed 58 kD NOSTRIN, but the protein level was significantly higher in women with PIH than in the normal group (P < 0.01). A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in placental tissue of women with PIH (r = -0.57, P < 0.01). It was concluded that the level of NOSTRIN expression in placenta of women with PIH was increased, which may play an important role in the pathogenesis of PIH.

Analysis of placental growth factor in placentas of normal pregnant women and women with hypertensive disorders of pregnancy.

To investigate the expressions of placental growth factor (PLGF) in placenta with hypertensive disorders of pregnancy (HDP), 45 women with HDP and 20 normally pregnant women were studied. Among 45 women with HDP, there were 23 cases of severe preeclampsia and one case of eclampsia. The location and level of PLGF proteins was determined by immunohistochemistry and Western blot. The expression of PLGF mRNA in placenta was assessed by reverse transcriptional-polymerase chain reaction (RT-PCR). The results showed that: (1) The distribution of PLGF in placenta with HDP was similar to normal one, which was mainly in the cytoplasm of villous syncytiotrophoblast and villous stroma; (2) The expression of PLGF protein was significantly decreased in placentas with mild and severe preeclampsia compared to the normal ones (0.3 +/- 0.4 vs 0.6 +/- 0.4, 0.2 +/- 0.5 vs 0.6 +/- 0.4, P < 0.01). There were no differences between the gestational hypertension placenta and normal one (0.5 +/- 0.6 vs 0.6 +/- 0.4, P > 0.05); (3) The transcription levels of the PLGF mRNA in placentas with preeclampsia were significantly lower than in normal groups (3.33 +/- 0.39 vs 4.87 +/- 0.60, 1.97 +/- 0.29 vs 4.87 +/- 0.60, P < 0.01), and no differences were found between the gestational hypertension placenta and normal groups. These findings suggest that the abnormal expression of PLGF in placentas is related to the pathogenesis of HDP.

Expression of Pin1 and Ki67 in cervical cancer and their significance.

In order to investigate the expression levels of Pin1 mRNA and protein in cervical cancer and its association with Ki67 and their clinical significance, amplification of Pin1 gene was examined by RT-PCR, and the expression of both Pin1 and Ki67 protein was detected by immunohistochemistry in cervical cancer tissues. It was shown that the expression levels of Pin1 were higher in cervical cancer than in normal cervical tissues (P < 0.05). The expression of Pin1 protein was increased progressively along with the disease process from normal cervix to CIN and to cervical cancer (P < 0.05). No significant difference in the Pin1 expression was found between disease stages (FIGO), pathological grades or pelvic lymph node metastasis status (P > 0.05). The expression of Pin1 was significantly higher in adenocarcinoma than in squamous carcinoma of the uterine cervix (P < 0.05). In cervical cancer, the overexpression of Pin1 was positively correlated with that of Ki67 (P < 0.05). These results suggested that the overexpression of Pin1 was closely related with cancer cell proliferation or progression of cervical cancer and contributed to oncogenesis. Pin1 may serve as a potential marker for cervical cancer diagnosis.

Analyses of Total Alkaloid Extract of Corydalis yanhusuo by Comprehensive RP × RP Liquid Chromatography with pH Difference.

A comprehensive two-dimensional (2D) reverse phase (RP) liquid chromatography (LC) method is developed for alkaloid analysis. This offline comprehensive 2D method is developed using different pH values. With a pH value of 10.5, most alkaloids appear in the form of neutral molecules possessing high retention factors based on their polarity, while the alkaloid polarity order is changed when the pH value decreased to 3.0. The performance of pH modulated 2D LC is demonstrated with 8 alkaloid standards which resulted in orthogonal separation. The developed method is then applied to total alkaloid separation in Corydalis yanhusuo. The first-dimension separation is carried out using methanol and water containing 1.0% ammonium hydroxide and a strong base-resistant RP column, which afforded a peak capacity of 94. The second-dimension analysis is carried out with a surface positive charge column providing a peak capacity of 205 using a mobile phase consisting of acetonitrile and water with 0.15% formic acid. 2D analyses of total alkaloid extract from C. yanhusuo afford a total peak capacity of 9090. Sixteen compounds were tentatively identified based on their ultraviolet spectrum and MS/MS analyses. The proposed method provides an alternative approach to achieve high peak capacity for analysis of alkaloid extract.

Arabidopsis NIP3;1 Plays an Important Role in Arsenic Uptake and Root-to-Shoot Translocation under Arsenite Stress Conditions.

In Arabidopsis, the nodulin 26-like intrinsic protein (NIP) subfamily of aquaporin proteins consists of nine members, five of which (NIP1;1, NIP1;2, NIP5;1, NIP6;1, and NIP7;1) were previously identified to be permeable to arsenite. However, the roles of NIPs in the root-to-shoot translocation of arsenite in plants remain poorly understood. In this study, using reverse genetic strategies, Arabidopsis NIP3;1 was identified to play an important role in both the arsenic uptake and root-to-shoot distribution under arsenite stress conditions. The nip3;1 loss-of-function mutants displayed obvious improvements in arsenite tolerance for aboveground growth and accumulated less arsenic in shoots than those of the wild-type plants, whereas the nip3;1 nip1;1 double mutant showed strong arsenite tolerance and improved growth of both roots and shoots under arsenite stress conditions. A promoter-ß-glucuronidase analysis revealed that NIP3;1 was expressed almost exclusively in roots (with the exception of the root tips), and heterologous expression in the yeast Saccharomyces cerevisiae demonstrated that NIP3;1 was able to mediate arsenite transport. Taken together, our results suggest that NIP3;1 is involved in arsenite uptake and root-to-shoot translocation in Arabidopsis, probably as a passive and bidirectional arsenite transporter.

The fronds tonoplast quantitative proteomic analysis in arsenic hyperaccumulator Pteris vittata L.

Pteris vittata, the first known arsenic hyperaccumulating plant, can accumulate very high concentration arsenic in its aboveground tissues, while low in roots. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the arsenic-hyperaccumulation in P. vittata, but the mechanism(s) of arsenic transport to vacuole are largely unknown. We obtained tonoplast isolated from fronds of P. vittata sporophyte grown under minus and 1mM arsenate for 3weeks by iodixanol step gradient centrifugation method, and then used TMPP protein labeling technology followed by liquid chromatography-a linear ion trap-Orbitrap hybrid mass spectrometer analysis for the quantitative detection of proteins. And we designed and used an "artificial" database for database searching. In total, 56 tonoplast proteins were identified; more than 70% of them were transport proteins. Under arsenate treatment, one TDT transporter protein, a member of the TerC family and a PDR-like protein were upregulated differentially. While V-ATPase subunits c, E, and G, and V-PPase, were downregulated. Additionally, the identified tonoplast proteins in our present study provide an informative basis for arsenic carriers or channels and help to clarify the regulation of tonoplast arsenic transport processes in P. vittata. BIOLOGICAL SIGNIFICANCE: Vacuole compartmentalization is crucial to As hyperaccumulator P. vittata, while there is limited known arsenic transport proteins involved in vacuole compartmentalization. In this paper, we obtained tonoplast of P. vittata fronds by iodixanol step gradient centrifugation method and then used TMPP protein labeling proteome technology for the quantitative detection of fronds tonoplast proteins. Our findings are the first challenge to the tonoplast proteins data mining of P. vittata which provide an informative basis for As carriers or channels. The proteomic approach in our study is suited for detecting alterations tonoplast protein and help to clarify the regulation of tonoplast transport processes. This article is part of a Special Issue entitled: Proteomics of non-model organisms.