P0859ACTIVATE ENDOGENOUS HYDROXYMETHYLATION TO RESTORE ERYTHROPOIETIN PRODUCTION IN FIBROTIC KIDNEYS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
AN JIE LUO ◽  
SHUEI-LIONG LIN
Keyword(s):  
Mrna Level ◽  
Dna Methyltransferases ◽  
The Body ◽  
Plasma Creatinine ◽  
Binding Motif ◽  
Murine Models ◽  
Coding Region ◽  
Kidney Fibrosis ◽  
Adult Mice ◽  
Targeting Vector

Abstract Background and Aims Our previous studies have shown that DNA methyltransferases (Dnmt) and Epo hypermethylation are upregulated in myofibroblasts in fibrotic murine kidneys. Demethylation leads to redifferentiation of myofibroblasts into pericytes. Furthermore, nontoxic doses of 5-azacytidine can restore EPO production and ameliorate anemia in mice with kidney fibrosis. Physiologic hydroxymethylation in zygotes is mediated by the 10 − 11 translocation enzymes (Tet1, Tet2, and Tet3). Method The mouse model of unilateral ureteral obstruction (UUO)-induced kidney fibrosis has been found to display enhanced Tet1 and Tet3 mRNA expression at day 7 and marked increase with the disease progression. Results According to the results, Tet expression significantly increased in fibrotic kidney. To study the effect of myofibroblast-specific Tet3 knockout in murine models of renal fibrosis. Tet3F/F mice were generated by a targeting vector with floxed region contains exons 8-9 of Tet3, which included the coding region for the conserved Fe2+-binding motif of dioxygenase. Gli1CreERT2/+;Tet3F/F mice were used to induce knockout of Tet3 in pericytes and myofibroblasts after tamoxifen administration. Tet3F/F mice were used as the control. Oral gavage of tamoxifen 8 mg/day for 3 days was used to activate Cre recombinase in Gli1+ pericytes and myofibroblasts in adult mice (6-8 week). After two-week washout period, mice were subjected to UUO for 7 or 14 days. The body weight, plasma creatinine and BUN levels showed no significant differences between groups in mice after induction and also UUO surgery. Plasma levels of BUN and creatinine were maintained within the normal range by normally functioning contralateral kidneys after UUO surgery. In UUO kidneys, knockout of Tet3 decreased renal α-SMA and Col1a1 expression. But plasma hematocrit and renal Epo mRNA level were not different between groups. In addition to Gli1CreERT2/+ mice we also used Pdgfrb-rtTATg;LC1Tg mice to breed with Tet3F/F mice for inducible knockout of Tet3. The plasma creatinine, BUN, and hematocrit were no different between groups after UUO surgery again. Conclusion Overall, these findings suggested increased Tet3 in myofibroblasts may play an important role in myofibroblast activation and fibrosis. Further studies need to explore the mechanisms.

Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

Diabetes ◽  
1994 ◽  
Vol 43 (10) ◽  
pp. 1234-1241 ◽  
Author(s):  
Y. H. Chen ◽  
L. Hansen ◽  
M. X. Chen ◽  
C. Bjorbaek ◽  
H. Vestergaard ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mrna Level ◽  
Regulatory Subunit ◽  
Coding Region

Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life

1994 ◽  
Vol 14 (12) ◽  
pp. 8219-8228
Author(s):  
P Belgrader ◽  
J Cheng ◽  
X Zhou ◽  
L S Stephenson ◽  
L E Maquat
Keyword(s):  
Triosephosphate Isomerase ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Coding Region ◽  
Protein Coding ◽  
Codon Recognition ◽  
Reverse Transcription Pcr ◽  
Nonsense Codon ◽  
Bona Fide ◽  
Nonsense Codons

Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination. Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutoff of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed.

scholarly journals Age-Related Alterations in the Behavior and Serotonin-Related Gene mRNA Levels in the Brain of Males and Females of Short-Lived Turquoise Killifish (Nothobranchius furzeri)

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1421
Author(s):  
Valentina S. Evsiukova ◽  
Elizabeth A. Kulikova ◽  
Alexander V. Kulikov
Keyword(s):  
Locomotor Activity ◽  
Body Mass ◽  
Mrna Level ◽  
Model Organism ◽  
The Body ◽  
Suitable Model ◽  
Mrna Levels ◽  
Age Related ◽  
Nothobranchius Furzeri ◽  
Males And Females

Short-lived turquoise killifish (Nothobranchius furzeri) have become a popular model organism for neuroscience. In the present paper we study for the first time their behavior in the novel tank diving test and the levels of mRNA of various 5-HT-related genes in brains of 2-, 4- and 6-month-old males and females of N. furzeri. The marked effect of age on body mass, locomotor activity and the mRNA level of Tph1b, Tph2, Slc6a4b, Mao, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes in the brains of N. furzeri males was shown. Locomotor activity and expression of the Mao gene increased, while expression of Tph1b, Tph2, Slc6a4b, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes decreased in 6-month-old killifish. Significant effects of sex on body mass as well as on mRNA level of Tph1a, Tph1b, Tph2, Slc6a4b, Htr1aa, 5-HT2a, Htr3a, Htr3b, Htr4, and Htr6 genes were revealed: in general both the body mass and the expression of these genes were higher in males. N. furzeri is a suitable model with which to study the fundamental problems of age-related alterations in various mRNA levels related with the brains 5-HT system.

Specific inhibition of response to purified protein antigens

1956 ◽  
Vol 146 (922) ◽  
pp. 18-33 ◽  
Keyword(s):  
Antibody Formation ◽  
The Body ◽  
Specific Inhibition ◽  
Protein Antigens ◽  
Bovine Albumin ◽  
Adult Mice ◽  
Immune Paralysis ◽  
Small Doses ◽  
Long Time ◽  
Stimulate Antibody

The injection of large doses of pneumococcus polysaccharides into adult mice fails to stimulate antibody formation and prevents subsequent small doses from stimulating antibody formation. This inhibition—termed immune paralysis (Felton 1949; Felton, Cameron & Prather 1941; Felton, Kauffmann, Prescott & Ottinger 1955) persists for at least 15 to 18 months. It is not possible to produce a similar effect with protein antigens. Dixon & Maurer (1953, I955 a,b ) injected very large quantities of bovine serum albumin into adult rabbits, 18 g/kg rabbit over a period of 6 weeks. Within 6 weeks of these injections four of the five animals produced antibody in response to injections of bovine albumin. It is not surprising that a long time elapsed before antibody could be detected, since during the persistence of antigen in the body, all antibody released would have combined with that vast excess of antigen.

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis

Development ◽  
2000 ◽  
Vol 127 (1) ◽  
pp. 129-141 ◽  
Author(s):  
L. Yan ◽  
K. Fei ◽  
J. Zhang ◽  
S. Dexter ◽  
M.P. Sarras
Keyword(s):  
Mrna Expression ◽  
The Body ◽  
Receptor Protein ◽  
Binding Motif ◽  
Immunofluorescence Studies ◽  
Basal Pole ◽  
Foot Process ◽  
Body Column ◽  
Endodermal Layer

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an “MAM” domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.

scholarly journals Differential expression of KvLQT1 and its regulator IsK in mouse epithelia

2001 ◽  
Vol 280 (2) ◽  
pp. C359-C372 ◽  
Author(s):  
Sophie Demolombe ◽  
Diego Franco ◽  
Piet de Boer ◽  
Sabina Kuperschmidt ◽  
Dan Roden ◽  
...  
Keyword(s):  
Slow Component ◽  
Genetic Disorder ◽  
Expression Patterns ◽  
Resting Potential ◽  
Delayed Rectifier ◽  
Coding Region ◽  
Epithelial Tissues ◽  
In Situ Data ◽  
Adult Mice

KCNQ1 is the human gene responsible in most cases for the long QT syndrome, a genetic disorder characterized by anomalies in cardiac repolarization leading to arrhythmias and sudden death. KCNQ1 encodes a pore-forming K+channel subunit termed KvLQT1 which, in association with its regulatory β-subunit IsK (also called minK), produces the slow component of the delayed-rectifier cardiac K+ current. We used in situ hybridization to localize KvLQT1 and IsK mRNAs in various tissues from adult mice. We showed that KvLQT1 mRNA expression is widely distributed in epithelial tissues, in the absence (small intestine, lung, liver, thymus) or presence (kidney, stomach, exocrine pancreas) of its regulator IsK. In the kidney and the stomach, however, the expression patterns of KvLQT1 and IsK do not coincide. In many tissues, in situ data obtained with the IsK probe coincide with β-galactosidase expression in IsK-deficient mice in which the bacterial lacZgene has been substituted for the IsK coding region. Because expression of KvLQT1 in the presence or absence of its regulator generates a K+ current with different biophysical characteristics, the role of KvLQT1 in epithelial cells may vary depending on the expression of its regulator IsK. The high level of KvLQT1 expression in epithelial tissues is consistent with its potential role in K+secretion and recycling, in maintaining the resting potential, and in regulating Cl− secretion and/or Na+absorption.

scholarly journals Localization of lysosomal acid phosphatase mRNA in mouse tissues.

1992 ◽  
Vol 40 (9) ◽  
pp. 1275-1282 ◽  
Author(s):  
C Geier ◽  
J Kreysing ◽  
H Boettcher ◽  
R Pohlmann ◽  
K von Figura
Keyword(s):  
Acid Phosphatase ◽  
Mrna Level ◽  
Pyramidal Cells ◽  
Housekeeping Gene ◽  
Lysosomal Acid ◽  
Tissue Sections ◽  
Adult Mice ◽  
Mouse Tissues ◽  
Major Species

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.

scholarly journals The Interconnections Between Somatic and Ovarian Aging in Murine Models

2020 ◽  
Author(s):  
Augusto Schneider ◽  
Tatiana D Saccon ◽  
Driele N Garcia ◽  
Bianka M Zanini ◽  
José V V Isola ◽  
...  
Keyword(s):  
Middle Age ◽  
Cellular Aging ◽  
The Body ◽  
Murine Models ◽  
Primordial Follicles ◽  
Ovarian Aging ◽  
Overwhelming Evidence ◽  
Theory Of Aging ◽  
Mammalian Female ◽  
Systemic Health

Abstract The mammalian female is born with a limited ovarian reserve of primordial follicles. These primordial follicles are slowly activated throughout the reproductive lifecycle, thereby determining lifecycle length. Once primordial follicles are exhausted, women undergo menopause, which is associated with several metabolic perturbations and a higher mortality risk. Long before exhaustion of the reserve, females experience severe declines in fertility and health. As such, significant efforts have been made to unravel the mechanisms that promote ovarian aging and insufficiency. In this review, we explain how long-living murine models can provide insights in the regulation of ovarian aging. There is now overwhelming evidence that most life-span–extending strategies, and long-living mutant models simultaneously delay ovarian aging. Therefore, it appears that the same mechanisms that regulate somatic aging may also be modulating ovarian aging and germ cell exhaustion. We explore several potential contributing mechanisms including insulin resistance, inflammation, and DNA damage—all of which are hallmarks of cellular aging throughout the body including the ovary. These findings are in alignment with the disposable soma theory of aging, which dictates a trade-off between growth, reproduction, and DNA repair. Therefore, delaying ovarian aging will not only increase the fertility window of middle age females, but may also actively prevent menopausal-related decline in systemic health parameters, compressing the period of morbidity in mid-to-late life in females.

scholarly journals Metabolic Acidosis Alters Expression of Slc22 Transporters in Mouse Kidney

2020 ◽  
Vol 45 (2) ◽  
pp. 263-274
Author(s):  
Janine Gottier Nwafor ◽  
Marta Nowik ◽  
Naohiko Anzai ◽  
Hitoshi Endou ◽  
Carsten A. Wagner
Keyword(s):  
Metabolic Acidosis ◽  
Mrna Level ◽  
The Body ◽  
Large Set ◽  
Protein Abundance ◽  
Waste Products ◽  
Anion Transporters ◽  
Solute Carrier ◽  
The Impact ◽  
Oxonic Acid

Introduction: The kidneys play a central role in eliminating metabolic waste products and drugs through transporter-mediated excretion along the proximal tubule. This task is mostly achieved through a variety of transporters from the solute carrier family 22 (SLC22) family of organic cation and anion transporters. Metabolic acidosis modulates metabolic and renal functions and also affects the clearance of metabolites and drugs from the body. We had previously shown that induction of metabolic acidosis in mice alters a large set of transcripts, among them also many transporters including transporters from the Slc22 family. Objective: Here we further investigated the impact of acidosis on Slc22 family members. Methods: Metabolic acidosis was induced for 2 or 7 days with NH4Cl, some animals also received the uricase inhibitor oxonic acid for comparison. Expression of transporters was studied by qPCR and immunoblotting. Results: NH4Cl induced no significant changes in plasma or urine uric acid levels but caused downregulation of Slc22a1 (Oct1), Slc22a6 (Oat1), Slc22a19 (Oat5), and ­Slc22a12 (Urat1) at mRNA level. In contrast, Slc22a4 mRNA (Octn1) was upregulated. On protein level, NH4Cl increased Octn1 (after 7 days) and Urat1 (after 2 days) abundance and decreased Oat1 (after 2 days) and Urat1 (after 7 days). Oxonic acid had no impact on protein abundance of any of the transporters tested. Conclusion: In summary, metabolic acidosis alters expression of several transporters involved in renal excretion of metabolic waste products and drugs. This may have implications for drug kinetics and clearance of waste metabolites.

Involvement of epidermal growth factor in inducing obesity in ovariectomized mice

1993 ◽  
Vol 265 (2) ◽  
pp. E323-E331 ◽  
Author(s):  
H. Kurachi ◽  
H. Adachi ◽  
S. Ohtsuka ◽  
K. Morishige ◽  
K. Amemiya ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Level ◽  
Subcutaneous Fat ◽  
Rabbit Antiserum ◽  
Weight Ratio ◽  
The Body ◽  
Ovariectomized Mice ◽  
Liver And Kidney ◽  
Epidermal Growth

Ovariectomy (Ovx) of mice significantly increases the epidermal growth factor (EGF) concentration in the submandibular gland. To elucidate the role of this elevated EGF in obesity of Ovx mice, we examined the effects of sialoadenectomy (Sx) and anti-EGF rabbit antiserum administration on the body weight (BW) gain and carcass fat deposition in Ovx animals. Studies were performed in four groups of mice consisting of control, Ovx, Ovx+Sx, and Ovx+anti-EGF groups. Ovx increased the BW gain compared with the control animals, whereas Sx and anti-EGF significantly reduced it. Although the relative weights (weight ratio to BW) of the liver and kidney were not significantly changed by Ovx, Sx, or anti-EGF treatment of Ovx mice, the relative weights of mesenteric, parametrial, and subcutaneous fat tissues were increased in Ovx mice, and this increase was significantly reduced by Sx or anti-EGF administration. Ovx induced adipocyte hypertrophy, and this effect was eliminated by Sx and anti-EGF. Moreover, acyl-CoA synthetase mRNA level was increased by Ovx, and this increase was reduced by Sx and anti-EGF in mesenteric fat tissue. These findings suggest that elevation of EGF may play a role in the induction of obesity in Ovx mice.

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