scholarly journals Strictosidine Synthase Coding Gene Expression towards Quinine Biosynthesis and Accumulation: Inconsistency in Cultured Cells and Fresh Tissues of Cinchona ledgeriana

2021 ◽  
Vol 26 (01) ◽  
pp. 131-138
Author(s):  
Diah Ratnadewi
Keyword(s):  
Gene Expression ◽  
Cultured Cells ◽  
Feedback Inhibition ◽  
Stem Bark ◽  
Strictosidine Synthase ◽  
Plant Materials ◽  
Young Leaves ◽  
Plant Alkaloids ◽  
Metabolites Production ◽  
Cinchona Ledgeriana

Strictosidine synthase, encoded by the gene STR, facilitates the regeneration of strictosidine, a critical intermediate for the synthesis of many plant alkaloids. The gene has, however, never been studied in Cinchona spp. The plants produce quinine alkaloid used for malaria medication, SARS-CoV-2 treatment and other industrial purposes. Cultured cells can produce the alkaloid but only at a much lower yield than the natural tree. This study describes STR expression and quinine content in various plant materials. Bioinformatic analyses were conducted on nine species of Rubiaceae to obtain reference sequences to design conservative primers for Cinchona ledgeriana STR (ClSTR). ClSTR expression was analyzed using qRT-PCR and quinine content was determined using HPLC. A complete coding sequence (CDS) of ClSTR was deposited in NCBI GenBank under the accession number MK422544.1. ClSTR was expressed in cultured cells, young and mature leaves, and stem bark. The elicited cells have higher expression than the control and they performed since the fourth week. However, the quinine content was greater in older cells. The gene expression in young leaves was superior, but quinine was most abundant in the stem bark. Every cell of C. ledgeriana, in culture or in the plant, expressed ClSTR and was capable of synthesizing the alkaloid quinine. The alkaloid from the leaves of the plant might be translocated and accumulated in the bark. No efflux of alkaloids from the confined cultured cells might contribute in triggering feedback inhibition in the biosynthetic pathway. This study revealed a critical obstacle in cell culture as a means of secondary metabolites production that needs further development of metabolic engineering. © 2021 Friends Science Publishers

scholarly journals Feedback Inhibition of Human Scavenger Receptor Class B Type I Gene Expression by Glucocorticoid in Adrenal and Ovarian Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (7) ◽  
pp. 3214-3224 ◽  
Author(s):  
Sofia Mavridou ◽  
Maria Venihaki ◽  
Olga Rassouli ◽  
Christos Tsatsanis ◽  
Dimitris Kardassis
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Feedback Inhibition ◽  
Steroid Hormone ◽  
Scavenger Receptor ◽  
Type I ◽  
Mrna Levels ◽  
Ovarian Cells ◽  
Scavenger Receptor Class ◽  
Class B

Scavenger receptor class B type I (SR-BI) facilitates the reverse transport of excess cholesterol from peripheral tissues to the liver via high-density lipoproteins. In steroidogenic tissues, SR-BI supplies cholesterol for steroid hormone production. We show here that the transcription of the human SR-BI gene is subject to feedback inhibition by glucocorticoid in adrenal and ovarian cells. SR-BI mRNA levels were increased in adrenals from corticosterone-insufficient Crh−/− mice, whereas corticosterone replacement by oral administration inhibited SR-BI gene expression in these mice. SR-BI mRNA levels were increased in adrenals from wild-type mice treated with metyrapone, a drug that blocks corticosterone synthesis. Experiments in adrenocortical H295R and ovarian SKOV-3 cells using cycloheximide and siRNA-mediated gene silencing revealed that glucocorticoid-mediated inhibition of SR-BI gene transcription requires de novo protein synthesis and the glucocorticoid receptor (GR). No direct binding of GR to the SR-BI promoter could be demonstrated in vitro and in vivo, suggesting an indirect mechanism of repression of SR-BI gene transcription by GR in adrenal cells. Deletion analysis established that the region of the human SR-BI promoter between nucleotides −201 and −62 is sufficient to mediate repression by glucocorticoid. This region contains putative binding sites for transcriptional repressors that could play a role in SR-BI gene regulation in response to glucocorticoid. In summary, this is the first report showing that glucocorticoid suppress SR-BI expression suggesting that steroidogenic tissues maintain steroid hormone homeostasis by prohibiting SR-BI-mediated high-density lipoprotein cholesterol uptake when the endogenous levels of glucocorticoid are elevated.

scholarly journals Markers for Gene Expression in Cultured Cells from the Nervous System

1972 ◽  
Vol 247 (10) ◽  
pp. 3159-3169
Author(s):  
Samuel H. Wilson ◽  
Bruce K. Schrier ◽  
John L. Farber ◽  
Edward J. Thompson ◽  
Roger N. Rosenberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Cultured Cells

Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression

1992 ◽  
Vol 12 (3) ◽  
pp. 1202-1208
Author(s):  
R A Graves ◽  
P Tontonoz ◽  
B M Spiegelman
Keyword(s):  
Gene Expression ◽  
Cultured Cells ◽  
Mutational Analysis ◽  
Cell Types ◽  
Specific Gene ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Specific Gene Expression ◽  
Nuclear Extracts ◽  
Adipogenic Gene

The molecular basis of adipocyte-specific gene expression is not well understood. We have previously identified a 518-bp enhancer from the adipocyte P2 gene that stimulates adipose-specific gene expression in both cultured cells and transgenic mice. In this analysis of the enhancer, we have defined and characterized a 122-bp DNA fragment that directs differentiation-dependent gene expression in cultured preadipocytes and adipocytes. Several cis-acting elements have been identified and shown by mutational analysis to be important for full enhancer activity. One pair of sequences, ARE2 and ARE4, binds a nuclear factor (ARF2) present in extracts derived from many cell types. Multiple copies of these elements stimulate gene expression from a minimal promoter in preadipocytes, adipocytes, and several other cultured cell lines. A second pair of elements, ARE6 and ARE7, binds a separate factor (ARF6) that is detected only in nuclear extracts derived from adipocytes. The ability of multimers of ARE6 or ARE7 to stimulate promoter activity is strictly adipocyte specific. Mutations in the ARE6 sequence greatly reduce the activity of the 518-bp enhancer. These data demonstrate that several cis- and trans-acting components contribute to the activity of the adipocyte P2 enhancer and suggest that ARF6, a novel differentiation-dependent factor, may be a key regulator of adipogenic gene expression.

Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells

1994 ◽  
Vol 14 (5) ◽  
pp. 3108-3114
Author(s):  
M H Baron ◽  
S M Farrington
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Targeted Mutagenesis ◽  
Erythroid Cell ◽  
Erythroid Cells

The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

scholarly journals Engineered External Guide Sequences Are Highly Effective in Inhibiting Gene Expression and Replication of Hepatitis B Virus in Cultured Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e65268 ◽  
Author(s):  
Zhigang Zhang ◽  
Gia-Phong Vu ◽  
Hao Gong ◽  
Chuan Xia ◽  
Yuan-Chuan Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cultured Cells ◽  
B Virus ◽  
Highly Effective

scholarly journals Assessment of NF-κB Inhibitor (SN50) Effect on Adipose Tumor Necrosis Factor-Alpha and Angiotensinogen Secretion and Expression

2021 ◽  
Author(s):  
Nasser M. Al-Dagheri ◽  
Assim A. Alfadda ◽  
Reem M. Sallam ◽  
Philip G. McTernan ◽  
Lotfi S. Bin Dahman
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Cultured Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Central Adiposity ◽  
Necrosis Factor Alpha ◽  
Hypertension Risk ◽  
Obese Subjects ◽  
Factor Alpha ◽  
Agt Gene

Central adiposity is one of the significant determinants of obesity-related hypertension risk, which may arise due to the abdominal fat depot's pathogenic inflammatory nature. Pro-inflammatory cytokines and adipokines up-regulation through nuclear factor-kappa B (NF-κB) activation in adipose tissue has been considered an essential function in the pathogenesis of obesity-related hypertension. This study aimed to ascertain the NF-κB inhibitor (SN50) effect on TNF-α and angiotensinogen (AGT) secretion and expression in mediating the anti-inflammatory effect through its impact on NF-κB activity in humans adipose tissue. Primary human adipocytes were isolated from 20 subjects among 10 overweight and 10 obese with and without hypertension and treated with 10ng/ml LPS in the presence and absence of NF-κB inhibitor, SN50 (50μg/ml). TNF-α secretion and NF-κB p65 activity were detected in supernatants extracted from cultured cells treated and untreated with LPS (10ng/ml) and SN50 (50μg/ml) using enzyme-linked immunosorbent assay (ELISA). The western blot technique detected the protein of NF-κB p65 and AGT. Gene expression of TNF-α and AGT was detected in cells and performed using quantitative real-time polymerase chain reaction (RT-PCR). Treatment of AbdSc adipocytes with LPS (10ng/ml) caused a significant increase in NF-κB p65 among overweight and obese subjects with and without hypertension (P= 0.001) at 24 hours incubation. In contrast, SN50-NF-κB inhibitor causes a reduction of NF-κB p65 in overweight (P= <0.001) and obese subjects with and without hypertension (P= 0.001) at 24 hours incubation. Treatment of AbdSc adipocytes with 10ng/ml LPS caused a significant increase in TNF-α secretion in overweight and obese subjects at all-time points (P= <0.001), whereas SN50 leads to a decrease in TNF-α secretion at 3 and 12 hours incubation. Treatment of AbdSc adipocytes with LPS (10ng/ml) caused increased TNF-α and AGT gene expression twofold compared with untreated cells, whereas, in the presence of SN50, it reduces mRNA AGT levels in both groups. Taken together, these adipokines with NF-κB activation may represent essential biomarkers to evaluate hypertension risk and to provide insight into the pathogenesis of obesity-related hypertension.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

scholarly journals Alkaloids Production and Cell Growth of Cinchona ledgeriana Moens: Effects of Fungal Filtrate and Methyl Jasmonate Elicitors

2021 ◽  
Vol 6 (1) ◽  
pp. 31-40
Author(s):  
Yustiny Andaliza Hasibuan ◽  
Diah Ratnadewi ◽  
Zainal Alim Mas’ud
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Secondary Metabolites ◽  
Methyl Jasmonate ◽  
Woody Plant ◽  
Cultured Cells ◽  
Cinchona Alkaloids ◽  
Cell Biomass ◽  
Cinchona Ledgeriana ◽  
Culture Technology

Cinchona alkaloids are known as antimalaria and anti-arrhythmic. Due to the long waiting time to harvest, cell culture technology is a challenge. This study aimed to determine the effects of elicitors, filtrate of two strains of endophytic fungi and methyl jasmonate (MeJA), in cell suspension culture of Cinchona ledgeriana on quinine and quinidine production. The cells were cultured for seven weeks in woody plant (WP) media treated with either of those elicitors in various concentrations. The cells growth was observed and the alkaloids were analyzed by HPLC. Cells treated with MeJA failed to grow that led to the cell biomass insufficiency for alkaloids determination.  It indicates that the cells are quite sensitive to even low concentration of MeJA that hampered the growth. Cells treated with the filtrate of Diaporthe sp. M13-Millipore filtered (S2M) gave the least cell biomass but presented the highest content of both alkaloids. Diaporthe sp. strain M-13 is stronger as elicitor than M-23 for this plant species. Filtrate of non-virulent fungi can elevate the biosynthesis of alkaloids. This reconfirms that cultured cells are capable to produce secondary metabolites and the productivity can be increased by using an appropriate elicitor.  

scholarly journals Expression of the Pseudorabies Virus Latency-Associated Transcript Gene during Productive Infection of Cultured Cells

1999 ◽  
Vol 73 (12) ◽  
pp. 9781-9788 ◽  
Author(s):  
Ling Jin ◽  
Gail Scherba
Keyword(s):  
Gene Expression ◽  
Pseudorabies Virus ◽  
Cultured Cells ◽  
Initiation Site ◽  
Early Gene ◽  
Productive Infection ◽  
Virus Latency ◽  
Infected Cells ◽  
Transcript Gene

ABSTRACT Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8.4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8.0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8.0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8′ and 8. Its synthesis was inhibited by cycloheximide but not by cytosine β-d-arabinofuranoside, which suggests that the 2.0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

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