Prenatal Glucocorticoid Administration Accelerates the Maturation of Fetal Rat Hepatocyte

Abstract Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because the liver is immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats.Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real time-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, HNF4α, HGF, Thy-1, cyclin B, and CDK1 in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production.The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration.The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in premature fetuses.

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Prenatal Glucocorticoid Administration Accelerates Maturation in the Hepatocytes of Fetal Rats

10.21203/rs.3.rs-235968/v2 ◽

2021 ◽

Author(s):

Tsukasa Kobayashi ◽

Yuko Takeba ◽

Yuki Ohta ◽

Masanori Ootaki ◽

Keisuke Kida ◽

...

Keyword(s):

Protein Production ◽

Cyclin B ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Rt Pcr ◽

Chain Reaction ◽

Low Birth Weight Infants ◽

Fetal Rats ◽

Polymerase Chain ◽

Hepatocyte Growth

Abstract Prenatal glucocorticoid (GC) is clinically administered to pregnant women at risk of preterm birth for maturation of the cardiopulmonary function. Preterm and low birth weight infants often experience liver dysfunction after birth because the liver is immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration regarding maturity of the liver in preterm rats. Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational day 17 and 19 before cesarean section. Real time polymerase chain reaction (RT-PCR) was then used to analyze the mRNA levels (albumin, HNF4α, HGF, Thy-1, cyclin B, and CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein production. Hepatocyte size enlarged because of growth and administration of prenatal DEX. Albumin, HNF4α, and hepatocyte growth factor (HGF) increased secondary to growth and administration of prenatal DEX. Cell cycle markers, cyclin B, and CDK1 gradually decreased during growth and by administration of DEX. These results suggest that prenatal GC administration achieves hepatocyte maturation via expression of HNF4α and HGF in premature fetuses.

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Prenatal Glucocorticoid Administration Accelerates Maturation in The Hepatocytes of Fetal Rats

10.21203/rs.3.rs-235968/v1 ◽

2021 ◽

Author(s):

Tsukasa Kobayashi ◽

Yuko Takeba ◽

Yuki Ohta ◽

Masanori Ootaki ◽

Keisuke Kida ◽

...

Keyword(s):

Cyclin B ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Hepatocyte Differentiation ◽

Rt Pcr ◽

Chain Reaction ◽

Low Birth Weight Infants ◽

Fetal Rats ◽

Polymerase Chain ◽

Hepatocyte Growth

Abstract Preterm and low birth weight infants often experience liver dysfunction after birth because the liver is immature. As such, prenatal glucocorticoid (GC) is clinically administered to pregnant women at risk of preterm birth for maturation of the cardiopulmonary function. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration regarding maturity of the liver in preterm rats. Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real time polymerase chain reaction (RT-PCR) was then used to analyze the mRNA levels (albumin, HNF4α, HGF, Thy-1, cyclin B, and CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein produced. Hepatocyte size enlarged because of growth and administration of prenatal DEX. Albumin, HNF4α, and hepatocyte growth factor (HGF) increased secondary to growth and administration of prenatal DEX. Cell cycle markers, Cyclin B, and CDK1 gradually decreased during growth and by administration of DEX. These results suggest that prenatal GC administration induces hepatocyte differentiation and achieves maturation via expression of HNF4α and HGF in premature fetuses.

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RFRP-3 synchronized with photoperiods regulates the seasonal reproduction of striped hamsters

Zygote ◽

10.1017/s0967199421000496 ◽

2021 ◽

pp. 1-7

Author(s):

Huiliang Xue ◽

Jinhui Xu ◽

Lei Chen ◽

Lei Zhao ◽

Ming Wu ◽

...

Keyword(s):

Follicle Stimulating Hormone ◽

Gonadotropin Releasing Hormone ◽

Neuron Activity ◽

Enzyme Linked Immunosorbent Assay ◽

Seasonal Reproduction ◽

Mrna Levels ◽

Rt Pcr ◽

Chain Reaction ◽

Long Day ◽

Polymerase Chain

Summary The purpose of this study was to investigate the effect of RFRP-3 synchronized with photoperiods on regulating the seasonal reproduction of striped hamsters. The striped hamsters were raised separately under long-day (LD; 16 h light/8 h dark), medium-day (MD; 12 h light/12 h dark) or short-day (SD; 8 h light/16 h dark) conditions for 8 weeks. RFRP-3 and gonadotropin-releasing hormone (GnRH) mRNA levels in the hypothalamus, testis or ovaries in three groups were detected using reverse transcription polymerase chain reaction (RT-PCR). Melatonin (MLT), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in serum were detected using enzyme-linked immunosorbent assay (ELISA). The correlation between RFRP-3 and GnRH mRNA and FSH and LH concentrations was also analyzed. MLT negatively regulated the expression of RFRP-3. Significant differences for RFRP-3 mRNA existed in the three groups, which positively correlated with the GnRH and the FSH and LH concentrations. RFRP-3 mRNA levels in the hypothalamus were significantly higher than those in ovaries or testis. RFRP-3 levels in the hypothalamus were significantly lower in female than in male under SD conditions, while those in ovaries were significantly higher than those in testes under LD conditions. MLT decreased RFRP neuron activity, and RFRP-3 regulated the reproduction of striped hamsters.

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Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽

10.1159/000517003 ◽

2021 ◽

pp. 1-6

Author(s):

Salman Khan ◽

Syed Asad Ali Shah ◽

Syed Muhammad Jamal

Keyword(s):

Polymerase Chain Reaction ◽

Viral Genome ◽

Foot And Mouth Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Kappa Value ◽

Polymerase Chain ◽

Pcr Assays ◽

Level Of Agreement

Background: Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. Methods: A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. Results: S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (p = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. Conclusions: The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

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Molecular cloning, expression analysis and developmental changes in ovarian follicles of goose 3β-hydroxysteroid dehydrogenase 1

Animal Production Science ◽

10.1071/an13315 ◽

2014 ◽

Vol 54 (8) ◽

pp. 992 ◽

Cited By ~ 3

Author(s):

Yingying Zhang ◽

Hehe Liu ◽

Mingjun Yang ◽

Shengqiang Hu ◽

Liang Li ◽

...

Keyword(s):

Adrenal Gland ◽

Follicular Development ◽

Hydroxysteroid Dehydrogenase ◽

Ovarian Follicles ◽

Mrna Levels ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Human And Mouse ◽

Main Factor

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase1 (3βHSD1) can catalyse the conversion of pregnenolone to progesterone in the △4-3-ketosteroid metabolic pathway. The aim of the present study was to clone 3βHSD1 and to determine whether this enzyme in the follicular wall has an effect on yolk progesterone in geese (Anser cygnoides). A putative coding sequence of 3βHSD1, which was 1134 nucleotides in length, was successfully obtained by using reverse transcription polymerase chain reaction (RT–PCR). A comparison of the deduced amino acid sequence with chicken, quail, zebra finch, cattle, horse, pig, human and mouse 3βHSD1 showed 89.7%, 88.4%, 87.3%, 55.6%, 54.0%, 53.5%, 55.3% and 52.9% similarity, respectively. The detection of 3βHSD1 mRNA levels in several tissues by quantitative real-time PCR showed that the highest level of 3βHSD1 was in the adrenal gland, followed by the ovary, which indicated that the gene we obtained was the adrenal gland/gonad-specific one. We measured the level of 3βHSD1 mRNA in the follicular wall and determined the concentration of progesterone in the yolk of these ovarian follicles; the concentration of progesterone in the yolk had a pattern of expression similar to that of 3βHSD1 in the follicular wall during follicular development. This result suggests that the expression of 3βHSD1 in the follicular wall may be a main factor that contributes to the accumulation of yolk progesterone.

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Ontogeny of erythropoietin gene expression in the sheep fetus: effect of dexamethasone at 60 days of gestation

Blood ◽

10.1182/blood.v84.2.460.bloodjournal842460 ◽

1994 ◽

Vol 84 (2) ◽

pp. 460-466

Author(s):

GB Lim ◽

K Jeyaseelan ◽

EM Wintour

Keyword(s):

Gene Expression ◽

Mrna Levels ◽

Rt Pcr ◽

Chain Reaction ◽

Fetal Plasma ◽

Liver And Kidney ◽

Polymerase Chain ◽

High Level ◽

A Site ◽

Sheep Fetus

We have used competitive reverse transcription and polymerase chain reaction (RT/PCR) to compare the levels of erythropoietin (Epo) mRNA in the liver and kidneys of the sheep fetus at 60, 80, 100, 130, and 140 days of gestation (term = 145 to 150 days). The effect of dexamethasone infusion in the ewe on Epo gene expression in the 60-day fetus was also investigated. Epo mRNA levels were highest at 60 days of gestation, the earliest age studied, in both liver and kidney. In the liver, Epo mRNA expression declined as gestation proceeded. Kidney Epo mRNA was maintained at a high level until 100 days of gestation, declining significantly in the 130-day fetus (P < .01). Treatment of ewes carrying 60-day fetuses with 0.76 mg/h dexamethasone for 48 hours resulted in a significant decrease in fetal plasma Epo values and Epo mRNA levels in both the liver and kidney. In the dexamethasone-treated fetuses, Epo mRNA in the liver was 52% of control values (P < .05), and in the kidney, 33% of control (P < .001). The results suggest that the kidney may play a more important role as a site of Epo synthesis in the early gestation sheep fetus than previously thought. Glucocorticoids may have a role in the regulation of Epo gene expression.

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Detection of small round structured viruses in clinical and environmental samples by polymerase chain reaction

Water Science & Technology ◽

10.2166/wst.1995.0644 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 375-382 ◽

Cited By ~ 8

Author(s):

M. Wolfaardt ◽

C. L. Moe ◽

W. O. K. Grabow

Keyword(s):

Polymerase Chain Reaction ◽

Tap Water ◽

Practical Method ◽

Glass Wool ◽

Polluted Water ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Elution Method

Norwalk (NV) and other small round structured viruses (SRSVs) have been identified as common causes of gastroenteritis. Outbreaks of Norwalk gastroenteritis have been associated with contaminated drinking water and food such as oysters and salads. The cloning and sequencing of the NV genome has made it possible to detect NV and related viruses by the reverse transcription-polymerase chain reaction (RT-PCR). We applied RT-PCR to detect SRSVs in faecal specimens from two gastroenteritis outbreaks in South Africa, designated “Christmas” and “Grootbrak” and were able to detect SRSVs in all of the three specimens from the Christmas outbreak and in two of 16 specimens from the Grootbrak outbreak. The RT-PCR procedure used appeared to be more sensitive for the detection of SRSVs in patient stool specimens than immune electron microscopy and NV antigen detection by enzyme linked immunosorbent assay. The RT-PCR procedure proved suitable for the detection of SRSVs in seeded samples of sewage, sewage sludge, river water, and tap water. However, sensitivity was lower for seeded samples of sewage and sludge than for tap water, which indicates interference by high levels of organic matter. The RT-PCR procedure was also used to show that small numbers of SRSVs can successfully be recovered from large volumes of water by means of a glass wool adsorption-elution method. Since no practical method is available for quantitation of the small numbers of SRSVs concerned, it was not possible to evaluate the efficiency of recovery. Although no SRSVs have been detected by direct testing of sewage and sludge samples, the results obtained in this study show that RT-PCR detection of SRSVs in sewage and polluted water environments is feasible, and that small numbers of the viruses can, like many other enteric viruses, successfully be recovered by means of a glass wool adsorption-elution method.

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RT-PCR Method for Detecting Cowpea Mottle Carmovirus in Vigna Germ Plasm

Plant Disease ◽

10.1094/pdis.1999.83.7.639 ◽

1999 ◽

Vol 83 (7) ◽

pp. 639-643 ◽

Cited By ~ 8

Author(s):

A. G. Gillaspie ◽

S. E. Mitchell ◽

G. W. Stuart ◽

R. F. Bozarth

Keyword(s):

Germ Plasm ◽

Enzyme Linked Immunosorbent Assay ◽

Mild Mosaic ◽

Rt Pcr ◽

Chain Reaction ◽

Viral Pathogens ◽

Highly Sensitive ◽

Pcr Method ◽

The Common ◽

Polymerase Chain

A highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was developed to detect cowpea mottle carmovirus (CPMoV) in newly acquired germ plasm of Vigna spp. It detected virus in tissues diluted up to 10-9. The preferred primers were designed from the RNA replicase cDNA sequence of CPMoV. These primers were able to detect CPMoV in plants infected with 10 different isolates of the virus. There were no cross-reactions with either bean mild mosaic or melon necrotic spot carmoviruses or any of the common cowpea viral pathogens tested. The RT-PCR method was up to 105 times more sensitive than direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA) in detecting CPMoV. The RT-PCR method gave no false positive reaction as is sometimes seen with ELISA.

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Circulation of bluetongue virus in goats in the Karamoja region of Uganda

Journal of the South African Veterinary Association ◽

10.4102/jsava.v84i1.922 ◽

2013 ◽

Vol 84 (1) ◽

Cited By ~ 1

Author(s):

Elijah N. Mulabbi ◽

Chrisostom Ayebazibwe ◽

Samuel Majalija ◽

Carrie A. Batten ◽

Christopher A.L. Oura

Keyword(s):

Real Time ◽

Whole Blood ◽

Bluetongue Virus ◽

Rna Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

High Seroprevalence ◽

Chain Reaction ◽

Polymerase Chain ◽

Karamoja Region

The presence of bluetongue virus (BTV) in indigenous goats from the Karamoja region of northern Uganda was investigated. A total of 300 goats were sampled (serum and whole blood) from five districts within the Karamoja region. The samples were analysed for the presence of bluetongue (BT) antibodies using a commercial Enzyme-linked immunosorbent assay (ELISA) and for the presence of BTV viral RNA by real-time Reverse transcription polymerase chain reaction (RT-PCR), because BTV is an RNA virus. Of the 300 goats tested, 269 (90%) were positive for BTV antibodies, indicating high levels of BTV circulation within the region. Out of the 150 whole blood samples tested for the presence of the virus by real-time RT-PCR, 84 (56%) were positive for BTV RNA. This study, which is the first of its kind in Uganda, showed a high seroprevalence of BT antibodies and active circulation of BTV in a high proportion of goats in the Karamoja region.

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Influence of mechanical and biological signals on gene expression in human MG-63 cells: evidence for a complex interplay between hydrostatic compression and vitamin D3 or TGF-β1 on MMP-1 and MMP-3 mRNA levels

Biochemistry and Cell Biology ◽

10.1139/o04-124 ◽

2005 ◽

Vol 83 (1) ◽

pp. 96-107 ◽

Cited By ~ 14

Author(s):

V Tasevski ◽

J M Sorbetti ◽

S S Chiu ◽

N G Shrive ◽

D A Hart

Keyword(s):

Gene Expression ◽

Vitamin D ◽

Polymerase Chain Reaction ◽

Bone Cells ◽

Mrna Levels ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Tgf Β1 ◽

In Cells

Biological mediators can influence the activity and differentiation of bone cells. 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) is known to induce differentiation of precursors into mature osteoblasts, and transforming growth factorβ1 (TGF-β1) can modulate the activity of bone cells leading to alterations in proliferation and gene expression patterns. Bone-derived cells were loaded via intermittent cyclic hydrostatic pressure (icHP) on cells under basal conditions and in the presence of 1,25-(OH)2D3 or TGF-β1. Evaluating the effects of loading on the cells allowed for a comparison to be made between responsiveness to biomechanical and biochemical stimuli and their potential interplay. The effects of icHP on mRNA levels for the specific genes involved in bone remodelling and differentiation were measured in MG-63 cells using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels for matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) were significantly, and uniquely, increased (p < 0.001) in cells exposed to icHP under serum-free conditions for 4–12 h. However, mRNA levels for MMP-3, but not MMP-1, were significantly enhanced in cells subjected to static hydrostatic pressure (HP). Treatment of cells with 1,25-(OH)2D3 resulted in increased (p < 0.001) mRNA levels for osteocalcin and decreased (p < 0.001) mRNA levels for both MMP-1 and MMP-3. In cells exposed to icHP and 1,25-(OH)2D3, the mRNA levels for both MMP-1 and MMP-3 were elevated (p < 0.001) compared with hormone alone, but not to the same degree (p < 0.01) as cells subjected to icHP alone. Addition of TGF-β1 to cells led to increases in cell proliferation and expression of collagen I, as well as decreases in expression of osteocalcin and MMP-1 and MMP-3. Exposure of cells to icHP and TGF-β1 again led to unique and significant increases in expression of MMP-1 and MMP-3. No changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or any of the other 9 genes assessed, including those for MMP-2 and MMP-13, were detected under any of the conditions described. Therefore, icHP can induce alterations in mRNA levels for a specific subset of genes in both premature and mature osteoblasts. Such stimuli can modulate the impact of potent biological mediators in defining patterns of gene expression by bone cells and potentially modify function in vivo.Key words: osteoblast, biomechanical loading,1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3), mRNA levels, reverse trans cription-polymerase chain reaction (RT-PCR), transforming growth factor-β1 (TGF-β1).

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