scholarly journals Molecular Mechanisms of Pollen-Pistil Interactions in Interspecific Crossing Barriers in the Tomato Family

2000 ◽  
Author(s):  
Yoram Eyal ◽  
Sheila McCormick
Keyword(s):  
Pollen Germination ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Dominant Negative ◽  
Peptide Sequence ◽  
Peptide Ligand ◽  
Hybrid Breakdown ◽  
Degenerate Primers

During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.

Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 921-929 ◽  
Author(s):  
Dong Chen ◽  
Audrey M. Bernstein ◽  
Paula P. Lemons ◽  
Sidney W. Whiteheart
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Dominant Negative ◽  
Dense Core ◽  
Protein Receptors ◽  
Dense Core Granules ◽  
Platelet Exocytosis ◽  
Granule Release

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant -SNAP and inhibited by a dominant negative -SNAP–L294A mutant or by anti–-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti–syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.

scholarly journals CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi47
Author(s):  
Marilin Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Justyna Przystal ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Profile ◽  
Transcriptional Networks ◽  
Molecular Alterations ◽  
Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

Expression Of Mutant Spliceosomal Protein SF3B1 Results In Dysregulated Hematopoietic Maturation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2773-2773
Author(s):  
Alexander C. Minella ◽  
Oscar Ramirez ◽  
Yanfei Xu ◽  
Tushar Murthy ◽  
Xiaodong Yang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Retroviral Vectors ◽  
Peptide Sequence ◽  
Causative Role ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Whole genome sequencing has recently revealed the prevalence of mutations in proteins directing splicing of RNA in up to half of the patients with Myelodysplastic Syndrome (MDS). Mutations in the protein SF3B1 are particularly common in MDS patients with the phenotypic abnormality termed ring sideroblasts (dysplastic erythroid precursors with perinculear rings formed by iron-laden mitochondria). The most common SF3B1 mutation in MDS patients results in a change from lysine to glutamic acid at amino acid position 700 (K700E). Given that splicing of RNA is a ubiquitous phenomenon, it is unclear how these mutations result in clonal proliferation and dysplastic hematopoiesis; two hallmark features of MDS. Furthermore, direct experimental evidence demonstrating a causative role for SF3B1 mutations in MDS-related phenotypes is lacking. To better understand how mutations of spliceosomal proteins contribute to MDS pathogenesis, we sought to define how expression of mutant SF3B1 changes erythroid maturation in vitro and in vivo. Native SF3B1 cDNA constructs are not amenable to bacterial propagation due to toxicity of its HEAT-domain repeats. We overcame this problem by codon optimization (changing the DNA sequence while preserving the native peptide sequence). Human cord blood derived CD34+ cells were transduced with retroviral vectors to express either the wild-type or K700E mutant of SF3B1. After a week of expansion in cytokines (IL-3, SCF and IL6), cells were induced to erythroid differentiation by addition of erythropoietin (EPO) and analyzed for surface markers of erythroid differentiation (CD 71, CD117, CD105, CD45 and CD235A) at regular intervals. K700E mutant expressing cells were found to have significantly reduced expression of CD105 when compared to wild-type SF3B1-expressing cells (average 50% recuction, n =8). CD105 or endoglin is a TGF-beta receptor accessory receptor expressed at high levels during intermediate stages of erythroid maturation. A more modest reduction of CD71 expression was also noted in K700E-SF3B1 cells. MDS bone marrow is known to express low levels of both CD105 and CD71 making our results clinically relevant. To further characterize how mutant SF3B1 may cause dysplastic hematopoiesis, we studied transduced and transplanted murine progenitor cells in vivo and in colony forming assays. Murine data demonstrate significantly reduced K700E-transduced hematopoietic progenitors (as defined by flow-cytometry) in vivo and impaired erythroid colony formation in vitro. Together, our results suggest that enforced expression of K700E-SF3B1 induces aberrant erythroid maturation and impairs homeostasis of hematopoietic precursor cells. Thus, we provide direct evidence that MDS-associated SF3B1 mutations perturb normal hematopoiesis and offer rationale for using our complementary experimental approach as a platform for elucidating the molecular mechanisms through which mutations in RNA splicing factors promote hematologic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Marilin Sophia Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Srinath Rajaraman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Subgroup ◽  
Replicative Stress ◽  
Helix Loop Helix ◽  
Molecular Events ◽  
Dominant Negative Variant

Abstract Background The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. Methods Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. Results The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. Conclusions Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

MO684A CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PD FLUIDS ON THE SECRETOME OF MESOTHELIAL AND ENDOTHELIAL CELLS

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Juan Manuel Sacnun ◽  
Rebecca Herzog ◽  
Klaus Kratochwill
Keyword(s):  
Endothelial Cells ◽  
Cardiovascular Risk ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Junctions ◽  
Peptide Ligand ◽  
Peritoneal Membrane

Abstract Background and Aims The composition of all currently available peritoneal dialysis fluids (PDF) triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels of the peritoneal membrane, eventually leading to technique failure. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of such additives on secretome-mediated signalling between cell-types of the peritoneal membrane which are relevant in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial (MC) and endothelial cells (EC) were co-cultured in transwell plates. MC were grown in the upper compartment and primary microvascular cells were grown in the lower compartment. MC were exposed to PDF with or without cytoprotective compounds (8 mM AlaGln in glucose-based PDF 3.86% or 10 mM LiCl in icodextrin-based PDF), while EC below were kept in medium. Cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release, neutral red uptake and cell morphology. Proteome and secretome profiles were analysed for both cell-types in co-culture or separately with an isobaric-tag labelling approach with a multiplexed liquid chromatography/mass spectrometry (LC-MS) approach. Prior to analysis of the secretome a bead-based equalizer approach based on a combinatorial peptide ligand library (CPLL) was performed to enrich low abundant proteins. Results EC injury after PD-fluid exposure of MC was decreased with the addition of AlaGln or LiCl, showing a link between the individual cell outcomes. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton re-organization, which characterize PDF cytotoxicity. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. We were able to identify and quantify 334 secreted proteins in the co-culture system. The secretome analysis showed variation in several clinically relevant proteins and important extracellular processes such as extracellular matrix reorganization, vesicle transport or collagen deposition. Comparison to previously published abundance profiles of omental arteriolar proteins from paediatric PD patient and age-matched controls confirmed overlapping protein regulation between endothelial cells in vitro and in vivo. Conclusion This study shows that harmful effects of PDF-stressed MC also affect EC and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the close proximity of different cell types in the peritoneal membrane for testing direct and indirect effects of cytoprotective additives. Characterisation of PD-induced perturbations may allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

scholarly journals Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown

2000 ◽  
Vol 347 (3) ◽  
pp. 653-660 ◽  
Author(s):  
Elaine A. POWERS ◽  
David P. THOMPSON ◽  
Peggy A. GARNER-HAMRICK ◽  
Wanxia HE ◽  
Anthony W. YEM ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Germinal Vesicle ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Glutathione S Transferase ◽  
Germinal Vesicle Breakdown ◽  
C Terminus

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.

Flavonoids as P-gp Inhibitors: A Systematic Review of SARs

2019 ◽  
Vol 26 (25) ◽  
pp. 4799-4831 ◽  
Author(s):  
Jiahua Cui ◽  
Xiaoyang Liu ◽  
Larry M.C. Chow
Keyword(s):  
Molecular Mechanisms ◽  
Metastatic Cancer ◽  
Drug Candidates ◽  
Chemical Structures ◽  
Transporter Family ◽  
Promising Area ◽  
New Generation ◽  
Nontoxic Inhibitors

P-glycoprotein, also known as ABCB1 in the ABC transporter family, confers the simultaneous resistance of metastatic cancer cells towards various anticancer drugs with different targets and diverse chemical structures. The exploration of safe and specific inhibitors of this pump has always been the pursuit of scientists for the past four decades. Naturally occurring flavonoids as benzopyrone derivatives were recognized as a class of nontoxic inhibitors of P-gp. The recent advent of synthetic flavonoid dimer FD18, as a potent P-gp modulator in reversing multidrug resistance both in vitro and in vivo, specifically targeted the pseudodimeric structure of the drug transporter and represented a new generation of inhibitors with high transporter binding affinity and low toxicity. This review concerned the recent updates on the structure-activity relationships of flavonoids as P-gp inhibitors, the molecular mechanisms of their action and their ability to overcome P-gp-mediated MDR in preclinical studies. It had crucial implications on the discovery of new drug candidates that modulated the efflux of ABC transporters and also provided some clues for the future development in this promising area.

Targeting PPARalpha in Alzheimer's Disease

2018 ◽  
Vol 15 (4) ◽  
pp. 345-354 ◽  
Author(s):  
Barbara D'Orio ◽  
Anna Fracassi ◽  
Maria Paola Cerù ◽  
Sandra Moreno
Keyword(s):  
Oxidative Stress ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Molecular Mechanisms ◽  
Redox Homeostasis ◽  
Antioxidant Response ◽  
Peroxisome Proliferator ◽  
Prevailing Paradigm

Background: The molecular mechanisms underlying Alzheimer's disease (AD) are yet to be fully elucidated. The so-called “amyloid cascade hypothesis” has long been the prevailing paradigm for causation of disease, and is today being revisited in relation to other pathogenic pathways, such as oxidative stress, neuroinflammation and energy dysmetabolism. The peroxisome proliferator-activated receptors (PPARs) are expressed in the central nervous system (CNS) and regulate many physiological processes, such as energy metabolism, neurotransmission, redox homeostasis, autophagy and cell cycle. Among the three isotypes (α, β/δ, γ), PPARγ role is the most extensively studied, while information on α and β/δ are still scanty. However, recent in vitro and in vivo evidence point to PPARα as a promising therapeutic target in AD. Conclusion: This review provides an update on this topic, focussing on the effects of natural or synthetic agonists in modulating pathogenetic mechanisms at AD onset and during its progression. Ligandactivated PPARα inihibits amyloidogenic pathway, Tau hyperphosphorylation and neuroinflammation. Concomitantly, the receptor elicits an enzymatic antioxidant response to oxidative stress, ameliorates glucose and lipid dysmetabolism, and stimulates autophagy.

Novel Findings of Anti-cancer Property of Propofol

2018 ◽  
Vol 18 (2) ◽  
pp. 156-165 ◽  
Author(s):  
Jiaqiang Wang ◽  
Chien-shan Cheng ◽  
Yan Lu ◽  
Xiaowei Ding ◽  
Minmin Zhu ◽  
...  
Keyword(s):  
General Anesthesia ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Intravenous Anesthetic ◽  
The Past ◽  
Anti Cancer ◽  
Underlying Mechanisms ◽  
Recent Attention

Background: Propofol, a widely used intravenous anesthetic agent, is traditionally applied for sedation and general anesthesia. Explanation: Recent attention has been drawn to explore the effect and mechanisms of propofol against cancer progression in vitro and in vivo. Specifically, the proliferation-inhibiting and apoptosis-inducing properties of propofol in cancer have been studied. However, the underlying mechanisms remain unclear. Conclusion: This review focused on the findings within the past ten years and aimed to provide a general overview of propofol's malignance-modulating properties and the potential molecular mechanisms.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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