This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

• Keywords
• DsRed, GFP, Plant cell pack, Plant expression vector, Tobacco BY-2 cells, Transient co-expression,
• MeSH
• Agrobacterium genetics   MeSH
• Cell Culture Techniques MeSH
• Red Fluorescent Protein MeSH
• Genetic Vectors * MeSH
• Plants, Genetically Modified genetics   MeSH
• Luminescent Proteins genetics   MeSH
• Protein Engineering methods   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Replicon MeSH
• Plant Cells metabolism   MeSH
• Nicotiana cytology   genetics   MeSH
• Green Fluorescent Proteins MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Luminescent Proteins MeSH
• Recombinant Proteins MeSH
• Green Fluorescent Proteins MeSH

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

• Keywords
• Cannabis sativa, GUS, Transient expression, Vacuum infiltration,
• MeSH
• Cannabis * genetics   metabolism   MeSH
• Gene Expression genetics   MeSH
• Plants, Genetically Modified * genetics   MeSH
• Glucuronidase genetics   metabolism   MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Plant * MeSH
• Genes, Reporter MeSH
• Nicotiana * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucuronidase MeSH

We have developed a Potato virus X (PVX)-based vector system compatible with the GoldenBraid 2.0 (GB) cloning strategy to transiently express heterologous proteins or peptides in plants for biotechnological purposes. This vector system consists of three domestication vectors carrying three GB parts-the cauliflower mosaic virus (CaMV) 35S promoter with PVX upstream of the second subgenomic promoter of the PVX coat protein (PVX CP SGP), nopaline synthase (NOS) terminator with PVX downstream of the first PVX CP SGP and the gene of interest (GOI). The full-length PVX clone carrying the sequence encoding a green fluorescent protein (GFP) as GOI was incorporated into the binary GB vector in a one-step reaction of three GB parts using the four-nucleotide GB standard syntax. We investigated whether the obtained vector named GFP/pGBX enables systemic PVX infection and expression of GFP in Nicotiana benthamiana plants. We show that this GB-compatible vector system can be used for simple and efficient assembly of PVX-based expression constructs and that it meets the current need for interchange of standard biological parts used in different expression systems.

• Keywords
• GoldenBraid, Nicotiana benthamiana, PVX vector, Potato virus X, transient expression,
• MeSH
• Genetic Vectors genetics   MeSH
• Potexvirus * genetics   MeSH
• Plants MeSH
• Nicotiana MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Green Fluorescent Proteins MeSH

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.

• Keywords
• Microscopy, Nicotiana benthamiana, Nicotiana tabacum, Phosphoinositides, Phospholipid-binding domains, Pollen tube, Transient expression,
• MeSH
• Gene Expression MeSH
• Fluorescent Dyes analysis   metabolism   MeSH
• Microscopy, Fluorescence methods   MeSH
• Phosphatidylinositols analysis   metabolism   MeSH
• Phospholipids analysis   metabolism   MeSH
• Microscopy, Confocal methods   MeSH
• Luminescent Proteins analysis   genetics   MeSH
• Pollen chemistry   genetics   MeSH
• Plant Cells chemistry   metabolism   MeSH
• Nicotiana chemistry   cytology   genetics   MeSH
• Transformation, Genetic MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Phosphatidylinositols MeSH
• Phospholipids MeSH
• Luminescent Proteins MeSH

In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells--expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

• MeSH
• 3T3 Cells MeSH
• RNA, Double-Stranded metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Immunoprecipitation MeSH
• Humans MeSH
• RNA, Small Interfering genetics   MeSH
• Mice MeSH
• Plasmids genetics   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Flow Cytometry MeSH
• Gene Expression Regulation genetics   MeSH
• Genes, Reporter genetics   MeSH
• Transfection methods   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Double-Stranded MeSH
• RNA, Small Interfering MeSH
• Protein Serine-Threonine Kinases MeSH

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.

• Publication type
• Journal Article MeSH

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

• Keywords
• Osmoregulation, Oxytocin, SON, TRPV1, Transgenic rats, Vasopressin,
• MeSH
• Action Potentials drug effects   MeSH
• HEK293 Cells MeSH
• Capsaicin analogs & derivatives   pharmacology   MeSH
• TRPV Cation Channels agonists   genetics   metabolism   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mannitol pharmacology   MeSH
• Neurons cytology   metabolism   MeSH
• Supraoptic Nucleus metabolism   MeSH
• Osmolar Concentration MeSH
• Oxytocin pharmacology   MeSH
• Rats, Transgenic MeSH
• Rats, Wistar MeSH
• Pyrazines pharmacology   MeSH
• Pyridines pharmacology   MeSH
• Temperature MeSH
• Calcium Signaling drug effects   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• capsazepine MeSH Browser
• Capsaicin MeSH
• TRPV Cation Channels MeSH
• Mannitol MeSH
• N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide MeSH Browser
• Oxytocin MeSH
• Pyrazines MeSH
• Pyridines MeSH
• Trpv1 protein, rat MeSH Browser

AIM: The pathogenesis of obesity has been associated with high intake of dietary fat, and some recent studies have explored the cellular mechanisms of oro-sensory detection of dietary fatty acids. We further assessed the role of transient receptor potential canonical (TRPC) channels in oro-sensory perception of dietary lipids. METHODS: We determined by RT-qPCR and western blotting the expression of TRPC3/6/7 channels in mouse fungiform taste bud cells (mTBC). Immunocytochemistry was used to explore whether TRPC3 channels were co-expressed with fatty acid receptors. We employed wild-type (WT) mTBC, and those transfected with small interfering RNAs (siRNAs) against TRPC3 or STIM1. Ca2+ signalling was studied in TBC from TRPC3-/- mice and their WT littermates. RESULTS: We demonstrate that mouse fungiform taste bud cells (mTBC) express TRPC3, but not TRPC6 or TRPC7 channels, and their inactivation by siRNA or experiments on TBC from TRPC3-/- mice brought about a decrease in fatty acid-induced gustatory Ca2+ signalling, coupled with taste bud CD36 lipid sensor. TRPC3 channel activation was found to be under the control of STIM1 in lingual mTBC. Behavioural studies showed that spontaneous preference for a dietary long-chain fatty acid was abolished in TRPC3-/- mice, and in mice wherein lingual TRPC3 expression was silenced by employing siRNA. CONCLUSION: We report that lingual TRPC3 channels are critically involved in fat taste perception.

• Keywords
• Ca2+ signalling, TRPC3 channels, fat taste, fatty acids, lipids,
• MeSH
• Taste Perception * MeSH
• Dietary Fats MeSH
• TRPC Cation Channels genetics   MeSH
• Lipids MeSH
• Mice MeSH
• Food Preferences * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dietary Fats MeSH
• TRPC Cation Channels MeSH
• Lipids MeSH
• TRPC3 cation channel MeSH Browser

Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar "Admiral", including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.

• MeSH
• Acyltransferases genetics   metabolism   MeSH
• Down-Regulation MeSH
• Gene Expression MeSH
• Humulus enzymology   genetics   virology   MeSH
• Plant Leaves enzymology   genetics   virology   MeSH
• RNA, Messenger chemistry   genetics   MeSH
• Plant Diseases virology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Propanols metabolism   MeSH
• Gene Expression Regulation, Enzymologic * MeSH
• Gene Expression Regulation, Plant MeSH
• RNA Interference MeSH
• RNA, Plant chemistry   genetics   MeSH
• RNA, Viral chemistry   genetics   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Plant Stems enzymology   genetics   virology   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Up-Regulation MeSH
• Viroids pathogenicity   physiology   MeSH
• Computational Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-phenylpropanol MeSH Browser
• Acyltransferases MeSH
• flavanone synthetase MeSH Browser
• RNA, Messenger MeSH
• Propanols MeSH
• RNA, Plant MeSH
• RNA, Viral MeSH
• Plant Proteins MeSH
• Transcription Factors MeSH

Hypoxic pulmonary vasoconstriction (HPV) is an important homeostatic mechanism in which increases of [Ca2+]i are primary events. In this study, primary cultured, human pulmonary artery smooth muscle cells (hPASMC) were used to examine the role of TRPC channels in mediating [Ca2+]i elevations during hypoxia. Hypoxia (PO2) about 20 mm Hg) evoked a transient [Ca2+]i elevation that was reduced by removal of extracellular calcium. Nifedipine and verapamil, blockers of voltage-gated calcium channels (VGCCs), attenuated the hypoxia-induced [Ca2+)]i elevation by about 30%, suggesting the presence of alternate Ca2+ entry pathways. Expression of TRPC1 and TRPC6 in hPASMC were found by RT-PCR and confirmed by Western blot analysis. Antagonists for TRPC, 2APB and SKF96365, significantly reduced hypoxia-induced [Ca2+]i elevation by almost 60%. Both TRPC6 and TRPC1 were knocked down by siRNA, the loss of TRPC6 decreased hypoxic response down to 21% of control, whereas the knockdown of TRPC1 reduced the hypoxia response to 85%, suggesting that TRPC6 might play a central role in mediating hypoxia response in hPASMC. However, blockade of PLC pathway caused only small inhibition of the hypoxia response. In contrast, AICAR, the agonist of AMP-activated kinase (AMPK), induced a gradual [Ca2+]i elevation, whereas compound C, an antagonist of AMPK, almost abolished the hypoxia response. However, co-immunoprecipitation revealed that AMPKalpha was not colocalized with TRPC6. Our data supports a role for TRPC6 in mediation of the [Ca2+]i elevation in response to hypoxia in hPASMC and suggests that this response may be linked to cellular energy status via an activation of AMPK.

• MeSH
• Pulmonary Artery cytology   physiology   MeSH
• Cell Hypoxia MeSH
• TRPC Cation Channels genetics   metabolism   MeSH
• TRPC6 Cation Channel MeSH
• Cells, Cultured MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• Myocytes, Smooth Muscle cytology   metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• AMP-Activated Protein Kinases metabolism   MeSH
• Muscle, Smooth, Vascular cytology   metabolism   MeSH
• Calcium metabolism   MeSH
• Calcium Channels metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• TRPC Cation Channels MeSH
• TRPC6 Cation Channel MeSH
• RNA, Small Interfering MeSH
• AMP-Activated Protein Kinases MeSH
• transient receptor potential cation channel, subfamily C, member 1 MeSH Browser
• TRPC6 protein, human MeSH Browser
• Calcium MeSH
• Calcium Channels MeSH