Steroid hormones often circulate in the blood as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues.
We have previously characterized STS activity in human and mouse breast and bone tissues, and we have shown that STS can provide estrogens to these tissues from circulating sulfated precursors.
This study was designed to characterize STS activity in a mouse fibroblast cell line (NIH-3T3). Using a radioactive estrone sulfate (E1S) conversion assay, we detected STS activity in cultured NIH-3T3 cells.
This activity was blocked by the STS inhibitors EMATE and STX-64, indicating authentic STS activity.
We also found that microsomes prepared from NIH-3T3 cells had relatively high STS activity and that cytosols had low activity, consistent with the known distribution of this enzyme to the endoplasmic reticulum.
Michaelis-Menten’s analysis of the NIH-3T3 microsomes indicated a Km of 10.9 µM using E1S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by 3H-E1S conversion assay, further supporting the conclusion that fibroblasts possess STS.
Furthermore, Western blotting confirmed the presence of immunoreactive STS in NIH-3T3 microsomes.
With regard to regulation, treatments of cultured NIH-3T3 cells revealed that cortisol and the synthetic glucocorticoids dexamethasone and prednisolone decreased STS activity, as we have found for cell lines from other tissues. The effect of cortisol was seen at both 10 µM and 1.0 µM but not at 0.1 µM.
Western blotting also indicated a decrease in STS immunoreactivity in cortisol-treated microsomes. The reduction in STS activity by dexamethasone in whole cells was rversed by the glucocorticoid receptor antagonist RU-486, indicating that glucocorticoid downregulation of STS activity is receptor mediated.
An inhibition assay on NIH-3T3 microsomes revealed that STS activity was inhibited significantly by 10 µM estradiol-17β, a known substrate inhibitor of E1S for STS, but not by 10 µM cortisol.
This is consistent with the idea that cortisol inhibits STS in NIH-3T3 cells through a regulatory mechanism rather than by substrate inhibition.
Our results could have important implications regarding local estrogen production by STS in fibroblasts, which are the most common connective tissue cells in the body, and on possible regulation of local estrogen levels by cortisol.
Transdermal permeation effect of collagen hydrolysates of deer sinew on <em>mouse</em> skin, ex vitro, and antioxidant activity, increased type I collagen secretion of percutaneous proteins in <em>NIH</em>/<em>3T3</em> cells.
The collagen hydrolysates as a cosmetic material have already been wide application. At present, few studies concern with transdermal behavior of collagen hydrolysates in vitro.
Deer sinew contains rich collagen with a content of 82.12%. Thus, this article mainly studies the transdermal effect of collagen hydrolysates of deer sinew (DSCH) on mouse skin, ex vitro, and to explore skincare protection of percutaneous proteins.
Collagen hydrolysates of deer sinew were extracted by 0.2% HCl and a two-step enzymatic method of pepsin-trypsin. The content of 17 amino acids of DSCH was detected by precolumn derivatization RP-HPLC.
Using Franz diffusion cell systems studied the transdermal effect of DSCH and then examined the percutaneous rate and molecular weight distribution of percutaneous proteins (PP).
Further, we studied the bioactivity of PP in vitro, such as the total antioxidant capacity and collagen secretion in NIH/3T3 cells.About 8.0% DSCH could penetrate skin of mouse, the molecular weight of PP mainly distributed in 5 ~ 13 kDa, accounted for 91.55%. Compared with the antioxidant activity of DSCH, PP had obvious antioxidant activity of scavenging radical cation.
Meanwhile, PP promoted cell proliferation and collagen I secretion in fibroblast cells; however, level of type III collagen has no change.Collagen hydrolysates of deer sinew may be used as cosmetic material to protect the skin from oxidative stress, to prevent premature skin aging.
CRISPR/Cas9 System for Efficient Genome Editing and Targeting in the <em>Mouse</em> <em>NIH</em>/<em>3T3</em> Cells.
Background
The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering.
In this study, the application of this system is reported for targeting Rag genes to produce mutant mouse NIH/3T3 cell line. The RagRagRag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID).
Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line.
Methods
Four single guide RNAs were designed to target sequences in the coding region of the RagRagRag1 and Rag2.
Results
Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in RagRagRagTherefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals.
Naringin prevents ultraviolet-B radiation-induced oxidative damage and inflammation through activation of peroxisome proliferator-activated receptor γ in <em>mouse</em> embryonic fibroblast (<em>NIH</em>-<em>3T3</em>) cells.
The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet-B (UVB) radiation (280-320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH-3T3).
In this study, 20 mJ/cm 2 of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH-3T3 cells.
Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH-3T3 cells.
Furthermore, naringin prevents UVB-induced mitogen-activated protein kinase families and nuclear factor-κB (NF-κB)-mediated activation of inflammatory factors, that is TNF-α, IL-6, IL-10, and COX-2 in NIH-3T3 cells. Peroxisome proliferator-activated receptor γ (PPARγ) is an anti-inflammatory agent and it suppressed the UVB-mediated oxidative and inflammatory responses.
In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH-3T3 cells. Thus, naringin prevents UVB-mediated inflammation and oxidative damage in NIH-3T3 cells probably over controlling NF-κB expression and activation of PPARγ.