scholarly journals Structure and Location Studies on Key Enzymes in Saponins Biosynthesis of Panax notoginseng

2019 ◽  
Vol 20 (24) ◽  
pp. 6121
Author(s):  
Pengguo Xia ◽  
Yujie Zheng ◽  
Zongsuo Liang
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Three Dimensional ◽  
Panax Notoginseng ◽  
Squalene Epoxidase ◽  
Protein Fusion ◽  
Active Components ◽  
Key Enzymes ◽  
Saponin Biosynthesis

Panax notoginseng is one of the most widely used traditional herbs for the treatment of various diseases, in which saponins were the main active components. At present, the research of P. notoginseng mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites of P. notoginseng. In our study, four coding sequences (CDS) encoding the key enzymes involved in saponin biosynthesis were cloned, namely farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarenediol-II synthase (DS), which contained open reading frame (ORF) of 1029 bp, 1248 bp, 1614 bp, and 2310 bp, and coded 342, 415, 537, and 769 amino acids, respectively. At the same time, their domains, secondary structures, three-dimensional structures, and phylogenetics trees were analyzed by kinds of bioinformatics tools. Their phylogenetics relationships were also analyzed. In addition, GFP (Green fluorescent protein) fusion genes were constructed by the plasmid transformation system to determine the subcellular localization. The results of subcellular localization showed that FPS, SE, and DS were mainly located in cytomembrane and its surrounding, while SS was located both in cytoplasm and cytomembrane. Our findings provided data demonstrating the expression patterns of genes involved in saponin biosynthesis and would facilitate efforts to further elucidate the biosynthesis of the bioactive components in P. notoginseng.

scholarly journals Key Enzymes of Triterpenoid Saponin Biosynthesis and the Induction of Their Activities and Gene Expressions in Plants

2010 ◽  
Vol 5 (7) ◽  
pp. 1934578X1000500 ◽  
Author(s):  
Chang Ling Zhao ◽  
Xiu Ming Cui ◽  
Yan Ping Chen ◽  
Quan Liang
Keyword(s):  
Medicinal Plants ◽  
Higher Plants ◽  
Squalene Epoxidase ◽  
Signal Pathways ◽  
Triterpenoid Saponin ◽  
Active Components ◽  
Gene Expressions ◽  
Triterpenoid Saponins ◽  
Key Enzymes ◽  
Saponin Biosynthesis

Triterpenoid saponins are one of the key active components of many medicinal plants. The biosynthetic pathway of triterpenoid saponins in higher plants and a lot of experimental results both indicated that the key enzymes involved in triterpenoid saponin synthesis are squalene synthase (SS), squalene epoxidase (SE), lupeol synthase (LS), dammarenediol synthase (DS), β-amyrin synthase (β-AS), cytochrome P450-dependent monooxygenase (PDMO), and glycosyltransferase (GT). The activities and coding genes of the key enzymes could be induced by a range of factors in various plant species. However, the effects of the factors on the content and composition of the triterpenoid saponins in specific plants are not certainly coincident, and different factors appear to induce the gene expressions of the key enzymes by different signal pathways and at different levels. This paper could provide a reference for strengthening the triterpenoid saponin-synthesizing capability of specific medicinal plants at enzyme and/or gene expression levels in order to improve the plants’ commercial values.

scholarly journals Global Analysis of Cell Type-Specific Gene Expression

2003 ◽  
Vol 4 (2) ◽  
pp. 208-215 ◽  
Author(s):  
David W. Galbraith
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Global Analysis ◽  
Expression Patterns ◽  
Three Dimensional ◽  
Global Gene Expression ◽  
Cell Types ◽  
Reasonable Assumption ◽  
Specific Gene ◽  
Different Cell Types

The tissues and organs of multicellular eukaryotes are frequently observed to comprise complex three-dimensional interspersions of different cell types. It is a reasonable assumption that different global patterns of gene expression are found within these different cell types. This review outlines general experimental strategies designed to characterize these global gene expression patterns, based on a combination of methods of transgenic fluorescent protein (FP) expression and targeting, of flow cytometry and sorting and of high-throughput gene expression analysis.

Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes

2010 ◽  
Vol 38 (6) ◽  
pp. 1681-1686 ◽  
Author(s):  
Ursula Eichenlaub-Ritter ◽  
Nora Staubach ◽  
Tom Trapphoff
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Maternal Age ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Model Organisms ◽  
Aged Mouse ◽  
Protein Fusion ◽  
Mouse Oocytes ◽  
Age Related

It has been known for more than half a century that the risk of conceiving a child with trisomy increases with advanced maternal age. However, the origin of the high susceptibility to nondisjunction of whole chromosomes and precocious separation of sister chromatids, leading to aneuploidy in aged oocytes and embryos derived from them, cannot be traced back to a single disturbance and mechanism. Instead, analysis of recombination patterns of meiotic chromosomes of spread oocytes from embryonal ovary, and of origins and exchange patterns of extra chromosomes in trisomies, as well as morphological and molecular studies of oocytes and somatic cells from young and aged females, show chromosome-specific risk patterns and cellular aberrations related to the chronological age of the female. In addition, analysis of the function of meiotic- and cell-cycle-regulating genes in oogenesis, and the study of the spindle and chromosomal status of maturing oocytes, suggest that several events contribute synergistically to errors in chromosome segregation in aged oocytes in a chromosome-specific fashion. For instance, loss of cohesion may differentially predispose chromosomes with distal or pericentromeric chiasmata to nondisjunction. Studies on expression in young and aged oocytes from human or model organisms, like the mouse, indicate that the presence and functionality/activity of gene products involved in cell-cycle regulation, spindle formation and organelle integrity may be altered in aged oocytes, thus contributing to a high risk of error in chromosome segregation in meiosis I and II. Genes that are often altered in aged mouse oocytes include MCAK (mitotic-centromere-associated protein), a microtubule depolymerase, and AURKB (Aurora kinase B), a protein of the chromosomal passenger complex that has many targets and can also phosphorylate and regulate MCAK localization and activity. Therefore we explored the role of MCAK in maturing mouse oocytes by immunofluorescence, overexpression of a MCAK–EGFP (enhanced green fluorescent protein) fusion protein, knockdown of MCAK by RNAi (RNA interference) and inhibition of AURKB. The observations suggest that MCAK is involved in spindle regulation, chromosome congression and cell-cycle control, and that reductions in mRNA and protein in a context of permissive SAC (spindle assembly checkpoint) predispose to aneuploidy. Failure to recruit MCAK to centromeres and low expression patterns, as well as disturbances in regulation of enzyme localization and activity, e.g. due to alterations in activity of AURKB, may therefore contribute to maternal age-related rises in aneuploidy in mammalian oocytes.

scholarly journals Luminescence lifetime imaging of three-dimensional biological objects

2021 ◽  
Vol 134 (9) ◽  
pp. 1-17
Author(s):  
Ruslan I. Dmitriev ◽  
Xavier Intes ◽  
Margarida M. Barroso
Keyword(s):  
Fluorescent Protein ◽  
Multiple Scales ◽  
Three Dimensional ◽  
Luminescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Protein Fusion ◽  
Phosphorescence Lifetime ◽  
Lifetime Imaging ◽  
Molecular Features ◽  
Biological Studies

ABSTRACT A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.

Identification and subcellular localization of a new cystinosin isoform

2008 ◽  
Vol 294 (5) ◽  
pp. F1101-F1108 ◽  
Author(s):  
Anna Taranta ◽  
Stefania Petrini ◽  
Alessia Palma ◽  
Liliana Mannucci ◽  
Martijn J. Wilmer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Human Kidney ◽  
Nephropathic Cystinosis ◽  
Protein Fusion ◽  
Lysosomal Compartment ◽  
Renal Tubular ◽  
Green Fluorescent ◽  
Carboxy Terminal

Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [35S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.

scholarly journals Profiling of Wheat Class III Peroxidase Genes Derived from Powdery Mildew-Attacked Epidermis Reveals Distinct Sequence-Associated Expression Patterns

2005 ◽  
Vol 18 (7) ◽  
pp. 730-741 ◽  
Author(s):  
Guosheng Liu ◽  
Xiaoyan Sheng ◽  
David L. Greenshields ◽  
Adam Ogieglo ◽  
Susan Kaminskyj ◽  
...  
Keyword(s):  
Powdery Mildew ◽  
Differential Expression ◽  
Fluorescent Protein ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Protein Fusion ◽  
Powdery Mildew Fungus ◽  
Mesophyll Cells ◽  
Signal Molecule

A cDNA library was constructed from leaf epidermis of diploid wheat (Triticum monococcum) infected with the powdery mildew fungus (Blumeria graminis f. sp. tritici) and was screened for genes encoding peroxidases. From 2,500 expressed sequence tags (ESTs), 36 cDNAs representing 10 peroxidase genes (designated TmPRX1 to TmPRX10) were isolated and further characterized. Alignment of the deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species demonstrated that these peroxidases fall into four distinct groups. Differential expression and tissue-specific localization among the members were observed during the B. graminis f. sp. tritici attack using Northern blots and reverse-transcriptase polymerase chain reaction analyses. Consistent with its abundance in the EST collection, TmPRX1 expression showed the highest induction during pathogen attack and fluctuated in response to the fungal parasitic stages. TmPRX1 to TmPRX6 were expressed predominantly in mesophyll cells, whereas TmPRX7 to TmPRX10, which feature a putative C-terminal propeptide, were detectable mainly in epidermal cells. Using TmPRX8 as a representative, we demonstrated that its C-terminal propeptide was sufficient to target a green fluorescent protein fusion protein to the vacuoles in onion cells. Finally, differential expression profiles of the TmPRXs after abiotic stresses and signal molecule treatments were used to dissect the potential role of these peroxidases in multiple stress and defense pathways.

Dysregulation of HOX as a Potential Therapeutic Target in Breast Cancer

2020 ◽  
Vol 27 ◽  
Author(s):  
Ji-Yeon Lee ◽  
Myoung Hee Kim
Keyword(s):  
Breast Cancer ◽  
Hox Genes ◽  
Therapeutic Potential ◽  
Expression Patterns ◽  
Genome Structure ◽  
Control Cell ◽  
Three Dimensional ◽  
Cellular Processes ◽  
Hox Protein

: HOX genes belong to the highly conserved homeobox superfamily, responsible for the regulation of various cellular processes that control cell homeostasis, from embryogenesis to carcinogenesis. The abnormal expression of HOX genes is observed in various cancers, including breast cancer; they act as oncogenes or as suppressors of cancer, according to context. In this review, we analyze HOX gene expression patterns in breast cancer and examine their relationship, based on the three-dimensional genome structure of the HOX locus. The presence of non-coding RNAs, embedded within the HOX cluster, and the role of these molecules in breast cancer have been reviewed. We further evaluate the characteristic activity of HOX protein in breast cancer and its therapeutic potential.

scholarly journals BanLec-eGFP Chimera as a Tool for Evaluation of Lectin Binding to High-Mannose Glycans on Microorganisms

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 180
Author(s):  
Zorana Lopandić ◽  
Luka Dragačević ◽  
Dragan Popović ◽  
Uros Andjelković ◽  
Rajna Minić ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Lectin Binding ◽  
Three Dimensional ◽  
Data Bank ◽  
Salmonella Typhi ◽  
Excitation Wavelength ◽  
Protein Data Bank Entry ◽  
High Mannose

Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in Escherichia coli. BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed β-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip®) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of Salmonella in flow cytometry, with especially pronounced binding to a Salmonella Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms.

Fluorescent protein expression in temperature tolerant and susceptible reef-building corals

2021 ◽  
pp. 1-10
Author(s):  
Exequiel Gabriel S. Dizon ◽  
Jeric P. Da-Anoy ◽  
Melissa S. Roth ◽  
Cecilia Conaco
Keyword(s):  
Spectral Properties ◽  
Coral Species ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Antioxidant Properties ◽  
Variable Expression ◽  
Acropora Digitifera ◽  
Insight Into

Abstract Fluorescent proteins (FPs) are reported to play an important role as photoprotectants and antioxidants in corals subjected to stressful conditions. Identifying the various FP genes expressed and FP gene expression patterns under stress in diverse coral species can provide insight into FP function. In this study, we identified 16 putative FP homologues from the transcriptomes of corals with varying susceptibility to elevated temperature, including Acropora digitifera, Favites colemani, Montipora digitata and Seriatopora caliendrum. Each coral expressed a different complement of FP transcripts, which were predicted to have distinct spectral properties. The most diverse and abundant repertoire of FP transcripts, including at least 6 green FPs, were expressed in the temperature-tolerant coral, F. colemani. In comparison, the other corals expressed fewer FP types. Specific FP transcripts exhibited variable expression profiles in coral fragments subjected to 32 ± 1 °C (treatment) or 28 ± 1 °C (control) for up to 72 h, suggesting that distinct FPs may have different roles. Further studies on the expression of the proteins encoded by these FP transcripts, their fluorescence activity, tissue localization, and possible antioxidant properties, are needed to reveal their contribution to thermal stress tolerance in certain species of corals.

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