249. Fibroblast growth factor receptor-1 (FGFR-1) is essential for spermiogenesis, capacitation and male fertility

2005 ◽  
Vol 17 (9) ◽  
pp. 99
Author(s):  
L. M. Cotton ◽  
G. M. Gibbs ◽  
D. M. De Kretser ◽  
M. K. O'Bryan
Keyword(s):  
Transgenic Mice ◽  
Male Fertility ◽  
Transgene Expression ◽  
Surrogate Marker ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Sperm Tail ◽  
Expression Levels ◽  
Over Expression ◽  
Sperm Development

Male infertility is often a result of irregular sperm development/function. The identification of snt-2 (Suc-1 associated Neurotrophic Factor Target 2) and Fgfr-1 to the sperm tail, lead to the hypothesis that Fgf signalling through snt-2 is involved in sperm tail development/function. To test this hypothesis, transgenic mice carrying a dominant-negative variant of Fgfr-1, driven by the protamine 1 promoter (haploid specific) were created. Breeding experiments confirmed male fertility; however, one line was significantly sub-fertile and demonstrated a significantly reduced daily sperm production (DSP, 30%↓). Transgene expression levels were up to 70 times above native mRNA levels in wt mice; however, there was a concurrent upregulation of the native receptor in transgenic mice, resulting in only a 6× over-expression in transgenic:native mRNA. To increase transgene expression, independent lines were crossed (double heterozygous, DH). DH transgene expression levels were up to 120 times above the native mRNA in wild type mice, resulting in a 20× over-expression in transgenic:native mRNA. Breeding experiments showed males from 1 cross were significantly subfertile with DSPs further reduced (41%↓). Collectively this data shows Fgfr-1 signalling is required for quantitatively normal spermiogenesis. Given the millions of sperm that mice produce, a 40%↓ in DSP is unlikely to be responsible for the sub-fertility observed i.e. 2 v. 9 pups/litter. Therefore, a disruption of Fgfr-1 signalling may also induce a post-testicular phenotype. Western blot analysis, using tyrosine phosphorylation as a surrogate marker of sperm capacitation, showed transgenic mice had a significantly attenuated ability to initiate capacitation. As capacitation is an absolute requirement for fertilisation, the absence of capacitating capability is probably the major contributor to the sub-fertility seen in the transgenic mice. This research demonstrates for the first time that the Fgfr-1 signalling cascade is one of several pathways associated with sperm development and function.

scholarly journals 257.Fibroblast growth factor receptor-1 (FGFR-1) is essential for spermiogenesis and male fertility

2004 ◽  
Vol 16 (9) ◽  
pp. 257
Author(s):  
L. M. Cotton ◽  
G. M. Gibbs ◽  
D. M. De Kretser ◽  
M. K. O'Bryan
Keyword(s):  
Transgenic Mice ◽  
Transgene Expression ◽  
Active Role ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Major Constituent ◽  
Sperm Function ◽  
Mrna Levels ◽  
Sperm Tail ◽  
Fgf Signalling

SNTs (Suc-1 associated Neurotrophic Factor Targets) are FGF signalling adaptors and crucial activators of the MAP/PI3 kinase pathways via FGFRs. Screening a rat testis library identified snt-2 as a potential ODF component. ODFs are a major constituent of the sperm tail that we hypothesise play an active role in motility. Using western blot analysis I have localised Fgfr-1 to the sperm tail. As such, I propose that Fgf signalling through snt-2 is involved in sperm tail development/function. To test this hypothesis, I created transgenic mice carrying a dominant-negative variant of Fgfr-1 driven by the protamine 1 promoter (haploid specific). Breeding experiments confirmed males were fertile, although one line showed a tendency towards reduced pup numbers. This effect was strengthened by Daily Sperm Production (DSP), showing significantly reduced DSP (30%↓) compared to wt mice. Transgene expression levels were expressed up to 70 times above native mRNA levels in wt mice; however there was a concurrent up-regulation of the native receptor in transgenic mice. Cumulatively this resulted in only a 6x over-expression in transgene: native mRNA, and illustrated the presence of a feedback mechanism controlling Fgfr-1 expression. To increase transgene expression, I crossed independent lines (double heterozygous, DH) males. Breeding experiments showed males from 1 cross were significantly subfertile (2 v. 10 in wt mice) . DSPs were further reduced, (41%↓) compared to wt mice. Collectively this data shows Fgfr-1 signalling is required for quantitatively normal spermiogenesis, but is also likely to have a post testicular role in sperm function. I hypothesise this is mediated via activation/regulation of motility through the MAP/PI3 kinase pathways. Further, these mouse models provide compelling evidence that infertility in Kallmann's Syndrome patients is composed of both hypothalamic and testicular components. These mice will provide valuable insights into the signal transduction mechanisms controlling sperm function and avenues for contraceptive development.

Abstract 310: Deoxycorticosterone Acetate (doca)-salt Exacerbates Hypertension and Vascular Dysfunction in Mice Expressing Dominant Negative Peroxisome Proliferator-activated Receptor-gamma (pparg) in Smooth Muscle

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Pimonrat Ketsawatsomkron ◽  
Deborah R Davis ◽  
Aline M Hilzendeger ◽  
Justin L Grobe ◽  
Curt D Sigmund
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Critical Role ◽  
Surrogate Marker ◽  
Dominant Negative ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor

PPARG, a ligand-activated transcription factor plays a critical role in the regulation of blood pressure and vascular function. We hypothesized that smooth muscle cell (SMC) PPARG protects against hypertension (HT) and resistance vessel dysfunction. Transgenic mice expressing dominant negative PPARG (S-P467L) in SMC or non-transgenic controls (NT) were implanted with DOCA pellet and allowed ad libitum access to 0.15 M NaCl for 21 days in addition to regular chow and water. Blood pressure was monitored by telemetry and mesenteric arterial (MA) function was assessed by pressurized myograph. At baseline, 24-hour mean arterial pressure (MAP) was similar between NT and S-P467L mice, while the transgenic mice were tachycardic. DOCA-salt increased MAP to a much greater degree in S-P467L mice (Δ MAP; S-P467L: +34.2±6.0, NT: +13.3±5.7, p<0.05 vs NT). Heart rate was similarly decreased in both groups after DOCA-salt. Vasoconstriction to KCl, phenylephrine and endothelin-1 did not differ in MA from DOCA-salt treated NT and S-P467L, while the response to vasopressin was significantly reduced in S-P467L after DOCA-salt (% constriction at 10-8 M, S-P467L: 31.6±5.6, NT: 46.7±3.8, p<0.05 vs NT). Urinary copeptin, a surrogate marker for arginine vasopressin was similar in both groups regardless of treatment. Vasorelaxation to acetylcholine was slightly impaired in S-P467L MA compared to NT at baseline whereas this effect was further exaggerated after DOCA-salt (% relaxation at 10-5 M, S-P467L: 56.1±8.3, NT: 79.4±5.6, p<0.05 vs NT). Vascular morphology at luminal pressure of 75 mmHg showed a significant increase in wall thickness (S-P467L: 18.7±0.8, NT: 16.0±0.4, p<0.05 vs NT) and % media/lumen (S-P467L: 8.4±0.3, NT: 7.1±0.2, p<0.05 vs NT) in S-P467L MA after DOCA-salt. Expression of tissue inhibitor of metalloproteinases (TIMP)-4 and regulator of G-protein signaling (RGS)-5 transcript were 2- and 3.5-fold increased, respectively, in MA of NT with DOCA-salt compared to NT baseline. However, this induction was markedly blunted in S-P467L MA. We conclude that interference with PPARG function in SMC leads to altered gene expression crucial for normal vascular homeostasis, thereby sensitizing the mice to the effects of DOCA-salt induced HT and vascular dysfunction.

scholarly journals Sex Differences in Estrogen-Dependent Transcription of Gonadotropin-Releasing Hormone (GnRH) Gene Revealed in GnRH Transgenic Mice

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3351-3358 ◽  
Author(s):  
Niren R. Thanky ◽  
Ruth Slater ◽  
Allan E. Herbison
Keyword(s):  
Transgenic Mice ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Transgene Expression ◽  
Gonadal Steroids ◽  
Critical Element ◽  
Sexually Dimorphic ◽  
Mrna Levels ◽  
Estrogen Replacement

Abstract The mechanisms through which gonadal steroids exert feedback actions on the activity of the GnRH neurons are not understood. Using a series of GnRH-LacZ transgenic mice we have examined the manner in which gonadal steroids suppress GnRH mRNA expression in male and female mice. The long-term gonadectomy of 5.5-GNZ-3.5 transgenic mice resulted in significant increases in cellular GnRH mRNA expression (P &lt; 0.05) and plasma LH concentrations (P &lt; 0.01) in both sexes. However, cellular levels of LacZ mRNA and β-galactosidase, which provide an index of GnRH gene transcription, were only elevated in males after gonadectomy. This sexually differentiated response was also observed in mice gonadectomized for 2 wk. Estrogen replacement in gonadectomized males returned transgene expression to intact levels. Experiments in transgenic mice with 3′ and 5′ deleted GnRH-LacZ constructs revealed that the suppressive influence of estrogen on LacZ transcription in the male required a critical element located between −5.2 and −1.7 kb of the GnRH promoter. These studies show that the suppression of GnRH mRNA expression by estrogen in the male involves a decrease in GnRH gene transcription that is dependent on a distal GnRH promoter element. The same mechanism does not exist in females, indicating that gonadal steroids suppress GnRH mRNA levels in a sexually dimorphic manner.

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1603-1610 ◽  
Author(s):  
Rikki R. Bharadwaj ◽  
Cecelia D. Trainor ◽  
Peter Pasceri ◽  
James Ellis
Keyword(s):  
Transgenic Mice ◽  
Transgene Expression ◽  
Double Mutant ◽  
Mobility Shift ◽  
Rich Region ◽  
Expression Levels ◽  
Integration Sites ◽  
High Level ◽  
Intron 2

Human β-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5′HS3 element requires the presence of the AT-rich region (ATR) in β-globin intron-2. Here, we examine the dependence of 5′HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5′HS3 β/γ-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P < .05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P < .05). We conclude that the ability of 5′HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5′HS3 β/γ-globin transgene expression and should be retained in LCRβ-globin expression cassettes designed for gene therapy.

Inactivation of the Mitotic Checkpoint Protein UBE2Q2 to Identify Its Function In Oncogenesis and Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5115-5115
Author(s):  
Nancy Wakeham ◽  
Sami Banerjee ◽  
David Frank Crawford
Keyword(s):  
Cell Cycle ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Mitotic Checkpoint ◽  
Skin Tumors ◽  
Regulatory Proteins ◽  
Checkpoint Activation ◽  
Tissue Specific ◽  
Checkpoint Protein ◽  
Over Expression

Abstract Abstract 5115 UBE2Q2 is a ubiquitin conjugating enzyme that was first identified in a microarray screen for mitotic regulatory proteins. Since UBE2Q2 is expressed in G2- and M-phases, we evaluated it as a possible regulator of mitotic entry or progression. While inactivation of the protein using either siRNA or dominant-negative protein expression caused no overt change in cell cycle progression in unperturbed cultured human cells, it induced a prolonged early mitotic arrest after cells were treated with vincristine or other microtubule inhibitors (MIs). In addition, inhibition of UBE2Q2 caused a pronounced and specific sensitization to the cytotoxicity of MIs, an effect mediated by apoptosis (Banerjee et al, Oncogene 26;6509-17). The cell cycle effect caused by UBE2Q2 inactivation was indistinguishable from that produced by over-expression of the checkpoint protein CHFR, suggesting that the two proteins function in the same mitotic checkpoint, the mitotic stress or prophase checkpoint. Although UBE2Q2 and CHFR appear to function in the same checkpoint, they do so in opposition to one another. In this study, we have explored possible mechanisms for UBE2Q2-mediated cell cycle arrest by assessing the effect of its inhibition on the accumulation of candidate gene products after checkpoint activation. We have found that prophase checkpoint activation in cells lacking functional UBE2Q2 is accompanied by accumulation of several mitotic regulatory proteins including Securin and Aurora-A. Studies are underway to determine if UBE2Q2 inactivation exerts this effect by inhibiting the ubiquitination of these proteins and to determine if proteins upregulated by UBE2Q2 inactivation are mediators of checkpoint function. Since CHFR functions as a tumor suppressor (Yu et al, Nat Genet 37;401-6) and in opposition to UBE2Q2, we hypothesize that UBE2Q2 will be oncogenic when constitutively over-expressed in vivo. In order to test this hypothesis, we have generated transgenic mice using an inducible system that allows expression of the FLAG-tagged UBE2Q2 transgene after Cre-mediated recombination, shown schematically in the accompanying figure. Several independent transgenic lines were generated and screened for expression of β-galactosidase (β-gal), the protein that serves as a marker for transgene expression. This system provides several advantages, allowing us to circumvent potential embryonic lethality in transgenics, providing an opportunity for tissue specific transgene expression, and allowing activation at a specific age or developmental stage. Two founders with abundant β-gal expression have been bred with mice expressing EIIa-Cre, which is constitutively expressed in all tissues. We have confirmed that Cre+-offspring from these matings have high level expression of UBE2Q2-FLAG. We are now breeding mice with the activated allele to generate a cohort of UBE2Q2-transgenic mice for evaluation. Since we hypothesize that UBE2Q2 over-expression will promote the development of malignancies, we will initially evaluate mice separately for the development of (1) spontaneous malignancies and (2) carcinogen-induced (DMBA) skin tumors. A group of about 100 transgenic mice and 100 littermate controls will be monitored for the spontaneous development of malignancies and submitted for necropsy in the case of death or an obvious malignancy. Since UBE2Q2 over-expression might have effects other than the promotion of tumors, we will also monitor the mice in these groups for weight gain (by weighing monthly), and metabolic and hematologic abnormalities (by laboratory evaluation). To assess the ability of UBE2Q2 over-expression to promote the development of DMBA-induced malignancies, we will give a single application of DMBA to the skin of about 25 transgenic mice and 25 littermate controls. Using this approach, there should be a low frequency of skin tumors in control mice (Yu et al, Nat Genet 37;401-6). In contrast, we expect a much higher frequency of skin tumors in UBE2Q2-transgenic mice. If UBE2Q2 over-expression promotes malignancies, we can evaluate the tissue specificity of this property by breeding with mice expressing a tissue-specific Cre. Finally, our data suggest that UBE2Q2 is a potentially useful target for the treatment of malignancies (Banerjee et al, Oncogene 26;6509-17). If our transgenic mice develop tumors, these mice will be a very useful tool for testing UBE2Q2 inhibitors that are developed. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human and Murine Osteocalcin Gene Expression: Conserved Tissue Restricted Expression and Divergent Responses to 1,25-Dihydroxyvitamin D3in Vivo

1997 ◽  
Vol 11 (11) ◽  
pp. 1695-1708 ◽  
Author(s):  
Natalie A. Sims ◽  
Christopher P. White ◽  
Kate L. Sunn ◽  
Gethin P. Thomas ◽  
Melanie L. Drummond ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Regulatory Sequences ◽  
Strontium Chloride ◽  
Mrna Levels ◽  
Low Calcium Diet ◽  
Osteocalcin Gene ◽  
Human Osteocalcin

Abstract Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3[ 1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6 ± 3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2 ± 0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2 ± 7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5 ± 0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly up-regulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.

In vivo expression profile of a H+-K+-ATPase α2-subunit promoter-reporter transgene

2004 ◽  
Vol 286 (6) ◽  
pp. F1171-F1177 ◽  
Author(s):  
Wenzheng Zhang ◽  
Xuefeng Xia ◽  
Lei Zou ◽  
Xiangyang Xu ◽  
Gene D. LeSage ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Distal Colon ◽  
Mrna Levels ◽  
Flanking Region

Because little is known about the molecular basis of transcriptional regulation of the murine H+-K+-ATPase α2 (HKα2) gene or other genes whose expression is restricted in part to the collecting duct, especially in vivo, we developed transgenic mice carrying an insertional HKα2 promoter-reporter gene construct. In these mice, the region −7,264/+253 of the HKα2 5′-flanking region controls expression of the reporter gene enhanced green fluorescent protein (EGFP). Patterns of HKα2/EGFP transgene expression were examined by fluorescence microscopy and immunoblotting. Of 10 major organs examined, EGFP immunoreactivity was detected abundantly in the kidney, and to a far lesser extent, in the brain and lung. Within the kidney, EGFP fluorescence was detected exclusively in the collecting ducts of transgenic mice and colocalized with the cellular distribution of both endogenous HKα2 and aquaporin-2, consistent with the known expression pattern of endogenous HKα2 in principal cells. Surprisingly, no transgene expression was evident by immunoblotting or fluorescence microscopy in the distal colon, the site of the highest endogenous HKα2 expression. Although previous studies of steady-state mRNA levels suggested differences in HKα2 gene regulation in the kidney and colon, our results provide the first direct evidence of differential transcriptional control of the HKα2 gene in these organs and suggest that regions outside the 5′-flanking region or other regulatory factors play a role in HKα2 expression in the distal colon.

A Novel Transgenic Mouse Model of Hyperfibrinogenemia

2001 ◽  
Vol 86 (08) ◽  
pp. 511-516 ◽  
Author(s):  
Alyssa Gulledge ◽  
Farhad Rezaee ◽  
Jan Verheijen ◽  
Susan Lord
Keyword(s):  
Mouse Model ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Northern Blots ◽  
Symptomatic Disease ◽  
Over Expression ◽  
Etiologic Role ◽  
Transgenic Lines ◽  
Models Of Disease ◽  
Risk Predictor

SummaryHyperfibrinogenemia is a risk predictor in several diseases, including cardiovascular disease. Nevertheless, it remains unknown whether elevated fibrinogen has an etiologic role in or is a reflection of disease pathogenesis, or both. To examine this question, we generated a mouse model of hyperfibrinogenemia. We isolated the mouse fibrinogen locus, containing the three fibrinogen genes, in a single P1 clone. This ~ 100 kb clone was injected into C57Bl/6J zygotes. Three transgenic lines were identified, two with elevated fibrinogen, 1.4- and 1.7-fold relative to normal. We characterized the line with the higher level. Northern blots of total RNA showed transgene expression was liver specific, and the message levels were 2- to 3-fold enhanced. Fibrinogen in transgenic mice was normal in both immunologic and clotting assays. Our data indicate that over-expression of all three fibrinogen genes is necessary to achieve hyperfibrinogenemia. We saw no increase in mortality or morbidity, no gross abnormalities in the organs, and no histologic differences in lung, liver, spleen or kidney, in transgenic mice relative to normal littermates. We conclude that elevated fibrinogen did not cause disease in mice. We anticipate that breeding these mice to other mouse models of disease will demonstrate whether hyper-fibrinogenemia has a role in the initiation or progression of symptomatic disease.

scholarly journals MicroRNA-513b-5p targets COL1A1 and COL1A2 associated with the formation and rupture of intracranial aneurysm

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zheng Zheng ◽  
Yan Chen ◽  
Yinzhou Wang ◽  
Yongkun Li ◽  
Qiong Cheng
Keyword(s):  
Cell Death ◽  
Intracranial Aneurysm ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Mrna Levels ◽  
Reporter Assay ◽  
Over Expression ◽  
Tnf Α

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

scholarly journals Methylobacterium oryzae Influences Isoepoxydon Dehydrogenase Gene Expression and Patulin Production by Penicillium expansum

Water ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1427
Author(s):  
Tiago Barros Afonso ◽  
Lúcia Chaves Simões ◽  
Nelson Lima
Keyword(s):  
Gene Expression ◽  
Distribution Systems ◽  
Water Distribution ◽  
Penicillium Expansum ◽  
Transcriptional Level ◽  
Positive Trend ◽  
Mrna Levels ◽  
Expression Levels ◽  
Production Values ◽  
Methylobacterium Oryzae

Biofilms can be considered the main source of microorganisms in drinking water distribution systems (DWDS). The ecology of a biofilm is dependent on a variety of factors, including the presence of microbial metabolites excreted by its inhabitants. This study reports the effect of the Gram-negative bacteria Methylobacterium oryzae on the idh gene expression levels and patulin production of Penicillium expansum mature biofilms. For this purpose, a RT-qPCR method to quantify idh mRNA levels was applied. In addition, the idh expression levels were compared with the patulin production. The results obtained revealed that the effect of the bacterium on pre-established P. expansum biofilms is dependent on the time of interaction. More mature P. expansum biofilms appear to be more resistant to the inhibitory effect that M. oryzae causes towards idh gene expression and patulin production. A positive trend was observed between the idh expression and patulin production values. The results indicate that M. oryzae affects patulin production by acting at the transcriptional level of the idh gene.

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