Our results highlight a hitherto unknown role for cortisol in acute stress adaptation that is nonspecific and involves changes in membrane fluidity. We demonstrate a rapid change in liver plasma membrane fluidity in response to stressed levels of cortisol in vitro. The fluidity changes seen with cortisol were not dose-related, but occurred above a certain threshold suggesting a receptor-independent mechanism likely associated with steroid incorporation into the lipid domain. This was supported by the inability of membrane impermeable ABT-199 Bcl-2 inhibitor cortisol-PEP to alter membrane fluidity. While changes in plasma membrane cholesterol levels alter lipid order that appears unlikely in the present case as membrane cholesterol remained unchanged in response to cortisol treatment. The cortisol-induced fluidization of liver plasma membrane appears to be steroid specific, as neither 17b-estradiol nor testosterone treatment showed a similar response in trout plasma membrane. This agrees with the recent findings that the chemical structure of the steroid backbone affect interaction with the lipid bilayer and subsequent changes in plasma membrane fluidity. It remains to be determined whether the membrane biophysical effect is also seen with other corticosteroids and not just cortisol. However, cortisol is the primary corticosteroid that is released into the circulation in response to stress in trout. The membrane fluidizing effect of cortisol seen in liver may be a generalized response affecting all tissues in response to stress. Mammalian studies reported a fluidizing effect of glucocorticoid on fetal rat liver and dog synaptosomal membranes, whereas an ordering effect was observed in rat renal brush border and rabbit cardiac muscle. This suggests that stress-mediated cortisol effect on membrane order may be tissue-specific, but this remains to be determined in fish. Altogether, our results indicate that stress-induced elevation in cortisol levels rapidly fluidizes liver plasma membrane in rainbow trout. AFM topographical and phase images further indicate that cortisol alters biophysical properties of liver plasma membranes. Specifically, cortisol exposure led to the reorganization of discrete microdomains, likely gel phase and disordered fluid-phase in the lipid bilayer. These discrete domains differed in height, which increased after cortisol treatment. A recent study on erythrocytes also reported a glucocorticoid-induced domain reorganization, which involved formation of large protein-lipid domains by hydrophobic and electrostatic interactions leading to alteration in membrane structure and elasticity. Similar domain changes have also been reported for synthetic lipids in response to halothane exposures or melting transitions, treatments that are known to increase membrane fluidity. Cortisol appears to have a greater effect on lower domains, as indicated by the greater change in surface adhesion following steroid treatment, compared to the higher lipid domains. Collectively, stressed levels of cortisol rapidly alter the biophysical properties of trout hepatic plasma membrane. We hypothesize that changes in membrane order by cortisol is the result of a non-uniform fluidization at the nanoscale among different membrane domains. Rapid changes to membrane order by cortisol may play a role in triggering acute stress-related signaling pathways. Indeed membrane order perturbations lead to rapid activation of cell signaling pathways, including protein kinases.
Month: November 2020
We genotyped this particular SNP rs895819 and assessed the association with risk of RCC in our ongoing case
Rates among females are generally about half of those among males. Though few risk factors are established for RCC, there are a number of predisposing conditions which are known to be related to the development of RCC, such as cigarette smoking, obesity, hypertension, diabetes, family history of cancer, and others. However, only a part of the individuals exposed to these risk factors will develop RCC in their life time, suggesting that individual differences including genetic susceptibility factors may be one of the most critical agents in renal cell carcinogenesis. MicroRNAs are a class of endogenous, small and non-coding RNAs, which are initially transcribed from genomic DNA to long primary transcripts and then are cleaved by nuclear Drosha into 60–70 nt hairpin-shaped precursor RNAs. Pre-miRNAs are exported to the cytoplasm by Exportin-5 and are further processed into,22 nt mature miRNA duplexes by the cleavage of Dicer. In association with RNA-induced silencing complex, miRNAs can induce mRNA degradation or translational repression by binding to the 39-untranslated region of their target genes at the posttranscriptional level. To date, it has been estimated that miRNAs modulate the expression of approximately 30% of human genes. MiRNAs are involved in a wide range of biological processes including cell cycle regulation, apoptosis and stem cell maintenance, development, metabolism and aging. It has been shown that miRNAs participate in human carcinogenesis as either tumor suppressors or oncogenes. Accumulative studies have suggested that single nucleotide polymorphisms or mutations could make a significant contribution to disease susceptibility and outcome. Genetic variants or mutations in miRNAs or pre-miRNAs may alter miRNA expression and/or maturation. One study has systematically identified 323 SNP in 227 known human miRNAs, and 12 SNPs are located within the miRNA precursors. The SNP rs895819 is located at the loop of premiR-27a and involves an A.G nucleotide transition. Sun et al. Temozolomide reported the polymorphism could lead to process variation, higher expression of miR-27a and eventually predisposition of gastric cancer. While Yang et al. found that G allele of rs895819 might impair the maturation of the miR-27a, thus, was associated with reduced familial breast cancer risk. Moreover, MertensTalcott et al. reported that in breast cancer cells, transfection of antisense miR-27a lead to increased expression of Zinc finger and BTB domain containing 10 and these responses were accompanied by decreased expression of Sp-dependent survival and angiogenic genes, including survivin, vascular endothelial growth factor, and VEGF receptor 1. However, over-expression of survivin was frequently observed in different types of cancer, including RCC. To date, there is no study on the association between pre-miR27a polymorphism and RCC susceptibility. Based on our knowledge regarding the new polymorphism and biological function of miR-27a, we hypothesized that the pre-miR27a polymorphism was associated with RCC susceptibility. To test this hypothesis, control study in a Chinese population. Nowadays, increasing studies have suggested that miRNAs, which play an important role in cancer progress as tumor oncogenes or tumor suppressors are involved in crucial biological processes, including development, differentiation, apoptosis and proliferation. Genetic variations in miRNAs have been reported to be related with many tumors, such as breast cancer, gastric cancer, colorectal cancer.
Which yielded an internal Met-free GFP sequence protein have recently been recognized as efficient ways to overcome the problems
In particular, an approach based on the methionine residue substitution method allows the efficient production of proteins with an N-terminal specific Paclitaxel functional group in vivo, which would pave the way to generate proteins with novel functions. The Met residue substitution method introduces unnatural Met analogues into a protein by reassigning the Met codon globally in a protein sequence. The simple procedure using the Met auxotroph enables the production of a range of proteins with functional groups on a large scale. Bio-orthogonally reactive groups, such as L-homopropargylglycine and L-azidohomoalanine, have been incorporated into the Met positions of proteins in vivo by adding the Met surrogates instead of Met because the wild-type Met-tRNA synthetase recognizes the unnatural amino acids. In addition, engineering of the substrate specificity of Met-tRNA synthetase can expand the scope of this methodology. Bacterial proteins are synthesized from Met and the removal process of the start Met can be suppressed by selecting the second residue next to the Met carefully. Therefore, Met analogues can be incorporated into the N-termini of proteins using the Met residue substitution method. However, the presence of the internal Met codons in the target sequences limits the successful application of the Met residue substitution method for N-terminal specific functionalization due to the reassignment of unnatural Met surrogates to internal Met codons as well as to the first Met codon. This problem can be overcome by engineering the protein sequence to be devoid of internal Met residues. Although this approach sometimes needs time-consuming protein engineering work to find internal Met-free variants having original functions of proteins, to our knowledge, this approach is the only one that makes the N-terminal specific modification of a protein possible. Our previous report showed that a protein sequence could be engineered to be an internal Met-free using a consensus-based concept. In the study, the internal Met residues of the single chain fragment variable antibody sequence were replaced successfully with other conserved amino acids without affecting the activity of the protein. This allowed subsequent N-terminal specific functionalization of the scFv using the Met residue substitution method. The stability of scFv probably contributed to the success of the approach because stability of a protein is known to be related to the resistance to mutations. However, it is easily expected that the Met removal based on consensus sequences may not always work, because most proteins are marginally stable and thus cannot withstand multiple changes in their sequences. In particular, hydrophobic residues such as Met are frequently located in the highly packed hydrophobic core, which makes it harder to generate functional Met-free protein sequences. We here engineered a green fluorescent protein to be an internal Met-free protein sequence and demonstrated its Nterminal functionalization using the in vivo Met residue substitution method. It was previously reported that mutations of the three Met residues in the core hydrophobic regions of GFP based on consensus approach induced complete misfolding of the protein. In the present study, a GFP devoid of internal Met residues was generated by semi-rational mutagenesis and its folding efficiency was improved by introducing mutations for GFP folding enhancement.