Identification of Nitric Oxide Responsive Genes in the Rudimentary Leaves of Litchi chinensis

Litchi (Litchi chinensis Sonn.) is an evergreen woody fruit tree widely cultivated in subtropical and tropical regions. Warm winter and hot spring often leads to abnormal floral differentiation in litchi. Under this condition, the rudimentary leaves in the floral buds expand and the inflorescences will stop developing. Thus, how to promote abortion of rudimentary leaves in litchi inflorescence are important for floral development. Previous study indicated that nitric oxide (NO) produced by sodium nitroprusside (SNP) promoted flowering and abortion of rudimentary leaves in litchi. In the present study, a suppression subtractive hybridization (SSH) was used to identify NO responsive genes. As a result, 16 high homologous ESTs were obtained from the SSH library of the SNP treated rudimentary leaves. The ESTs were classified into three groups. They are disease/defensive, protein destination and storage, and protein synthesis. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that 6 out of the 7 randomly selected ESTs’expression showed an increasing trend from 0 h to 10 h of SNP treatment. It is suggested that the litchi homologs 18S ribosomal RNA gene, cytochrome P450 like TBP, and the senescence-associated protein, chaperone protein, and a hypothetical protein encoding genes may be involved in the NO-induced senescence in litchi rudimentary leaves. LcERD15-like may be a key gene involved in this process.

Download Full-text

Nitric Oxide-Mediated Induction of Dispersal inPseudomonas aeruginosaBiofilms Is Inhibited by Flavohemoglobin Production and Is Enhanced by Imidazole

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01832-17 ◽

2017 ◽

Vol 62 (3) ◽

Cited By ~ 2

Author(s):

Xinyi Zhu ◽

Hyun-Suk Oh ◽

Yu Chiu Beverly Ng ◽

Pei Yi Peggy Tang ◽

Nicolas Barraud ◽

...

Keyword(s):

Nitric Oxide ◽

Control Strategies ◽

Bacterial Species ◽

Pcr Analysis ◽

Signal Molecule ◽

Content Type ◽

Reverse Transcription Pcr ◽

Biofilm Dispersal ◽

New Perspective ◽

Underlying Mechanisms

ABSTRACTThe biological signal molecule nitric oxide (NO) was found to induce biofilm dispersal across a range of bacterial species, which led to its consideration for therapeutic strategies to treat biofilms and biofilm-related infections. However, biofilms are often not completely dispersed after exposure to NO. To better understand this phenomenon, we investigated the response ofPseudomonas aeruginosabiofilm cells to successive NO treatments. When biofilms were first pretreated with a low, noneffective dose of NO, a second dose of the signal molecule at a concentration usually capable of inducing dispersal did not have any effect. Amperometric analysis revealed that pretreatedP. aeruginosacells had enhanced NO-scavenging activity, and this effect was associated with the production of the flavohemoglobin Fhp. Further, quantitative real-time reverse transcription-PCR (qRT-PCR) analysis showed thatfhpexpression increased by over 100-fold in NO-pretreated biofilms compared to untreated biofilms. Biofilms of mutant strains harboring mutations infhporfhpR, encoding a NO-responsive regulator offhp, were not affected in their dispersal response after the initial pretreatment with NO. Overall, these results suggest that FhpR can sense NO to trigger production of the flavohemoglobin Fhp and inhibit subsequent dispersal responses to NO. Finally, the addition of imidazole, which can inhibit the NO dioxygenase activity of flavohemoglobin, attenuated the prevention of dispersal after NO pretreatment and improved the dispersal response in older, starved biofilms. This study clarifies the underlying mechanisms of impaired dispersal induced by repeated NO treatments and offers a new perspective for improving the use of NO in biofilm control strategies.

Download Full-text

Identification and Characterization of a Repeat-in-Toxin Gene Cluster in Vibrio anguillarum

Infection and Immunity ◽

10.1128/iai.01308-07 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2620-2632 ◽

Cited By ~ 48

Author(s):

Ling Li ◽

Jessica L. Rock ◽

David R. Nelson

Keyword(s):

Atlantic Salmon ◽

Gene Cluster ◽

Vibrio Anguillarum ◽

Hypothetical Protein ◽

Pcr Analysis ◽

Toxin Gene ◽

Reverse Transcription Pcr ◽

Hemolysin Gene ◽

Negative Mutant

ABSTRACT Vibrio anguillarum is the causative agent of vibriosis in fish. Hemolysins of V. anguillarum have been considered virulence factors during infection. One hemolysin gene, vah1, has been previously identified but does not account for all hemolytic activity. The mini-Tn10Km mutagenesis performed with a vah1 mutant resulted in a hemolysin-negative mutant. The region surrounding the mutation was cloned and sequenced, revealing a putative rtx operon with six genes (rtxACHBDE), where rtxA encodes an exotoxin, rtxC encodes an RtxA activator, rtxH encodes a conserved hypothetical protein, and rtxBDE encode the ABC transporters. Single mutations in rtx genes did not result in a hemolysin-negative phenotype. However, strains containing a mutation in vah1 and a mutation in an rtx gene resulted in a hemolysin-negative mutant, demonstrating that the rtx operon is a second hemolysin gene cluster in V. anguillarum M93Sm. Reverse transcription-PCR analysis revealed that the rtxC and rtxA genes are cotranscribed, as are the rtxBDE genes. Additionally, Vah1 and RtxA each have cytotoxic activity against Atlantic salmon kidney (ASK) cells. Single mutations in vah1 or rtxA attenuate the cytotoxicity of V. anguillarum M93Sm. A vah1 rtxA double mutant is no longer cytotoxic. Moreover, Vah1 and RtxA each have a distinct cytotoxic effect on ASK cells, Vah1 causes cell vacuolation, and RtxA causes cell rounding. Finally, wild-type and mutant strains were tested for virulence in juvenile Atlantic salmon. Only strains containing an rtxA mutation had reduced virulence, suggesting that RtxA is a major virulence factor for V. anguillarum.

Download Full-text

MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway

Open Medicine ◽

10.1515/med-2020-0198 ◽

2020 ◽

Vol 15 (1) ◽

pp. 1202-1212

Author(s):

Aichun Zhang ◽

Yangzi Jin

Keyword(s):

Allergic Rhinitis ◽

Inflammatory Cells ◽

Lavage Fluid ◽

Specific Ige ◽

Inflammatory Factors ◽

Pcr Analysis ◽

Reverse Transcription Pcr ◽

Pathway Activation ◽

Nasal Lavage Fluid

AbstractAllergic rhinitis (AR) is one of the most common chronic diseases. This study examined whether microRNA (miR)-182-5p plays a role in AR by regulating toll-like receptor 4 (TLR4). First, data demonstrated that TLR4 was a target of miR-182-5p. Subsequently, AR mouse model was established to explore the role of miR-182-5p and TLR4 in AR in vivo. Initially, quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that miR-182-5p was downregulated, while TLR4 expression was upregulated in AR mice. Then we found that miR-182-5p mimic reduced the frequency of sneezing and nose rubbing of the AR mice. In addition, miR-182-5p mimic significantly increased ovalbumin (OVA)-specific IgE and leukotriene C4 expression levels in nasal lavage fluid (NLF) and serum of AR mice. miR-182-5p mimic decreased the number of inflammatory cells in NLF of AR mice. It also reduced the levels of inflammatory factors in the serum of AR mice, such as interleukin (IL)-4, IL-5, IL-13, IL-17 and tumor necrosis factor (TNF)-α, while increasing the release of IFN-γ and IL-2. Finally, miR-182-5p mimic inhibited NF-κB signaling pathway activation in AR mice. However, all effects of miR-182-5p mimic on AR mice were reversed by TLR4-plasmid. In conclusion, miR-182-5p/TLR4 axis may represent a novel therapeutic target for AR.

Download Full-text

Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6541-6549.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6541-6549 ◽

Cited By ~ 19

Author(s):

Gilbert Thierry Lamothe ◽

Thierry Putallaz ◽

Han Joosten ◽

Joey D. Marugg

Keyword(s):

Reverse Transcription ◽

Membrane Filtration ◽

Limit Of Detection ◽

Mineral Waters ◽

Pcr Analysis ◽

Rna Sequences ◽

Natural Mineral ◽

Reverse Transcription Pcr ◽

Viral Particles ◽

Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

Download Full-text

The effects of quercetin on the expression of SREBP-1c mRNA in high-fat diet-induced NAFLD in mice

Journal of Basic and Clinical Physiology and Pharmacology ◽

10.1515/jbcpp-2020-0423 ◽

2021 ◽

Vol 32 (4) ◽

pp. 637-644

Author(s):

Jamal Nasser Saleh Al-maamari ◽

Mahardian Rahmadi ◽

Sisca Melani Panggono ◽

Devita Ardina Prameswari ◽

Eka Dewi Pratiwi ◽

...

Keyword(s):

High Fat Diet ◽

Fatty Liver Disease ◽

Inhibitory Effect ◽

Pcr Analysis ◽

Regulator Gene ◽

High Fat ◽

Alcoholic Fatty Liver ◽

Reverse Transcription Pcr ◽

Hematoxylin Eosin ◽

Alcoholic Fatty Liver Disease

Abstract Objectives The study aimed to determine the effect of quercetin on the expression of primary regulator gene involved in lipogenesis and triglycerides synthesis in the liver, and the sterol regulatory binding protein-1c (SREBP-1c) mRNA in non-alcoholic fatty liver disease (NAFLD) with a high-fat diet (HFD) model. Methods Fifty-six Balb/c mice were divided into seven groups: standard feed; HFD; HFD and quercetin 50 mg/kg for 28 days; HFD and quercetin 100 mg/kg BW for 28 days; HFD and quercetin 50 mg/kg for 14 days; HFD and quercetin 100 mg/kg for 14 days; HFD and repaired fed for 14 days. Quercetin was administered intraperitoneally. The animals were sacrificed 24 h after the last treatment; the liver was taken for macroscopic, histopathological staining using hematoxylin–eosin and reverse transcription-PCR analysis sample. Results HFD significantly increased the expression of SREBP-1c mRNA; meanwhile, quercetin and repaired feed significantly reduced the expression of SREBP-1c mRNA in the liver. Quercetin at a dose of 50 mg/kg and 100 mg/kg also improved liver cells’ pathological profile in high-fat diet NAFLD. Conclusions The present study suggests that quercetin has an inhibitory effect on SREBP-1c expression and improved liver pathology in NAFLD mice.

Download Full-text

Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

Journal of Virology ◽

10.1128/jvi.74.21.10176-10186.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10176-10186 ◽

Cited By ~ 31

Author(s):

T. Yamaguchi ◽

S. L. Kaplan ◽

P. Wakenell ◽

K. A. Schat

Keyword(s):

Cell Line ◽

Fibroblast Cell ◽

Northern Blotting ◽

Marek's Disease ◽

Pcr Analysis ◽

Marek’S Disease ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Herpesvirus Of Turkeys ◽

Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

Download Full-text

miR-369-3p serves as prognostic factor and regulates cancer progression of hepatocellular carcinoma

Personalized Medicine ◽

10.2217/pme-2020-0012 ◽

2021 ◽

Author(s):

Can Chen ◽

Yi Zong ◽

Jiaojiao Tang ◽

Ruisheng Ke ◽

Lizhi Lv ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Cancer Progression ◽

Cell Counting ◽

Migration And Invasion ◽

Pcr Analysis ◽

Reverse Transcription Pcr ◽

Huh7 Cells

Background: The aim of this study was to investigate the role of miR-369-3p in hepatocellular carcinoma (HCC). Materials & methods: The expression levels of miR-369-3p were detected using the quantitative real-time reverse transcription-PCR analysis. The cell counting kit-8 and transwell assays were used to explore the effects of miR-369-3p on cell proliferation, migration and invasion of HCC cells. Results: The miR-369-3p expression was downregulated in HCC tissues and cell lines, in comparison to the normal controls, respectively. In vitro, overexpression of miR-369-3p in Hep 3B and Huh7 cells inhibited cell proliferation, migration and invasion. SOX4 was a direct target of miR-369-3p. Conclusion: Our results suggested that miR-369-3p may be a tumor suppressor in HCC by targeting SOX4.

Download Full-text

Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in Apple

10.21203/rs.2.21019/v3 ◽

2020 ◽

Author(s):

Xiya Zuo ◽

Shixiang Wang ◽

Wen Xiang ◽

Huiru Yang ◽

Muhammad Mobeen Tahir ◽

...

Keyword(s):

Gene Family ◽

Expression Profiles ◽

Expression Patterns ◽

Floral Transition ◽

Bimolecular Fluorescence Complementation ◽

Pcr Analysis ◽

Transcriptional Responses ◽

Genome Database ◽

Reverse Transcription Pcr ◽

Conserved Core

Abstract Background: Apple (Malus domestica Borkh.) is one of the most popular cultivated fruit crops in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple.Results: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14‑3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus.Conclusion: We identified the Md14-3-3s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3s in floral transition.

Download Full-text

Amelioration of C - Reactive Protein and Lectin Like Oxidised Low Density Lipoprotein Receptor Complex Induced Endothelial Dysfunction by Oligomeric Proanthocyanidins.

10.21203/rs.3.rs-770001/v1 ◽

2021 ◽

Author(s):

Sankar Jamuna ◽

Rathinavel Ashokkumar ◽

Niranjali Devaraj Sivasithamparam

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Dysfunction ◽

Capillary Tube ◽

Morphological Changes ◽

Protein Docking ◽

Pcr Analysis ◽

Inhibitory Mechanism ◽

C Reactive Protein ◽

Reactive Protein

Abstract C-reactive protein (CRP) is a well established biochemical marker for atherosclerosis. Inflammation induced by CRP promotes endothelial dysfunction. Modification of LDL inside the artery wall favors the elevation of this acute phase protein. The mechanism of OxLDL+CRP complex is unrevealed so far. Hence, this mechanism was considered as the important factor to trigger the monocyte to macrophages differentiation which in leads to foam cells formation. Hence this key event should be targeted and focused on how this complex (OxLDL+CRP) proceeds to endothelial dysfunction. OPC is a well known cardioprotective flavon-3-ols. The present study is challenged between the protective roles of OPC against the deleterious effect of this complex (OxLDL+CRP) on endothelial cells. Monolayer of Endothelial cells were incubated with THP-1 monocytes for 48 h supplemented with OxLDL (10mg/ml) + CRP (10 mg/ml) complex and treated with OPC (100mg/ml). Morphological changes, cell migration assay and capillary tube forming assay was carried out. Myeleoperoxidase levels were estimated to determine the adhesion of monocytes onto EC monolayer. RT-PCR analysis of L-Selectin was done. The quantification of NO levels and analysis of mRNA expressions of eNOS is to determine the nitric oxide demand caused due to OxLDL+CRP complex. LOX-1, scavenger receptor levels were analysed by mRNA expression. Proinflammatory markers such as IL-6, MCP-1 and IL-1b were studied. Accumulation of ROS levels were measured fluorimetrically using DCF-DA. Spectrophotometric analysis of Sirius red dye binding collagen levels was observed. Mitochondrial membrane potential was determined by JC-1 dye and cell cycle analysis was done by FACS analysis. Protein –Protein docking was carried out between CRP and LOX-1. This docked protein complex were again docked with OPC and atrovastatin to show the inhibitory mechanism of CRP binding with LOX-1. OPC showed a promising inhibitory mechanism against OxLDL+CRP complex. To emphasis the results OPC treated group showed decreased levels of proinflammatory markers, LOX-1 and L-Selectin levels. Endothelial nitric oxide levels were increased upon OPC treatment and reduction in the ROS levels. Endothelial cells apoptosis was prevented by OPC. Docking studies showed that in the absence of ligands (OPC) binding of CRP and LOX-1 was greater and vice versa in the presence of ligands. To conclude, OxLDL + CRP complex inhibitory effects of OPC could maintain the normal homeostasis.

Download Full-text

Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in Apple

10.21203/rs.2.21019/v5 ◽

2020 ◽

Author(s):

Xiya Zuo ◽

Shixiang Wang ◽

Wen Xiang ◽

Huiru Yang ◽

Muhammad Mobeen Tahir ◽

...

Keyword(s):

Gene Family ◽

Expression Profiles ◽

Expression Patterns ◽

Economic Value ◽

Floral Transition ◽

Bimolecular Fluorescence Complementation ◽

Pcr Analysis ◽

Transcriptional Responses ◽

Genome Database ◽

Reverse Transcription Pcr

Abstract Background: Apple (Malus domestica Borkh.) is a popular cultivated fruit crop with high economic value in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple.Results: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14‑3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus.Conclusion: We identified the Md14-3-3s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3s in floral transition.

Download Full-text