Author: small molecule

However, in cell culture, it is extremely difficult to establish cell networks that mimic in vivo systems. As a result, a safe margin has been applied to health risk assessments to take into consideration the possibility of insufficient evaluation, particularly regarding interspecies differences, though such extrapolation to humans using safe margins occasionally results in overestimation of risks. However, the underestimation of risks by a small safety margin exposes humans to significant danger. Therefore, to perform more accurate health risk assessments, the development of an in vivo evaluation system that can reproduce human responses to toxic factors would be an important breakthrough. For many years, mouse models transgenically expressing human genes or harboring transplanted human cells, tissues, and organs, called humanized mice, have been developed to reproduce the responses of human cells in vivo. Mice that are humanized by transplantation of human cells are able to establish networks of human cells in their bodies. The available diverse mouse models were developed by transplantation of various types of cells to immunodeficient strains of mice. In cancer research, the biology of human tumor growth, metastasis, and angiogenesis has been evaluated in these mouse models. More recently, by transplanting human hepatocytes into liver-failure immunodeficient mice, mice with human livers have been developed for the study of human infectious diseases and metabolism. Moreover, allowing for the establishment of a functional human-like hematopoietic lineage. These techniques have proven valuable for the in vivo study of human hematopoietic stem cell function, infectious disease, and drug discovery, among other research questions. Interspecies differences in responses to toxicants are influenced greatly by the specificity and expression pattern of receptors, metabolic enzymes, and many other Rapamycin molecules. A human-like hematopoietic lineage may mimic the response to toxicants by human cells, and such humanized mice may therefore prove to be powerful tools for health assessment and aid in our evaluation of the hematotoxicity of various factors, while accounting for interspecies differences. Hematotoxicity is evaluated according to many factors, including decreased hematopoietic cell counts, abnormal blood coagulation, aberrant myelopoiesis, and induction of leukemia, all of which can be caused by diverse risk factors. Toxicants, such as benzene, can differentially affect human or animal hematopoietic lineages. Here, we took advantage of mice harboring a human-like hematopoietic lineage as a tool for assessing human hematotoxicity in vivo. These mice were established by transplanting NOG mice with human CD34+ cells. The response to benzene, a model toxicant, was measured by determining decreases in the number of leukocytes. Furthermore, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice. To evaluate whether the response to benzene by Hu-NOG mice reflected interspecies differences, the degrees of benzene-induced hematotoxicities in Mo-NOG and Hu-NOG mice were compared. Here, we evaluated the toxic response of a human-like hematopoietic lineage established in NOG mice using the hematotoxicant benzene. Benzene-induced hematotoxicity is known to be transmitted by the aryl hydrocarbon receptor. Benzene metabolism is mediated by signals transmitted through interactions between AhR and benzene, benzene metabolites, or both, and the resulting benzene metabolites and reactive oxygen species induce cell damage. In hematopoietic cells, the AhR is expressed selectively by immature cells, such as hematopoietic stem/progenitor cells. Therefore, the toxic response of immature cells is the main cause of benzene-induced hematotoxicity.

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.

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.

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.

Some of them showed robust CAD association, SNP association showed inconsistent magnitudes of CAD association. It is assumed that such inconsistency is explained by pleiotropic actions of the lipid loci in part. In the present study, to test associations between lipid traits and CAD for SNPs from 22 candidate loci recently reported by GWA studies and their meta-analyses in populations of European descent, we performed a replication study in Japanese populations. From the viewpoint of population genetics, generalization of lipid association results previously identified in European GWA studies to non-European populations is an issue of interest, because it can facilitate the fine mapping of common causal variants by providing clues to whether SNPs identified in European GWA studies are simply tag-SNP or “synthetic association” markers or are more likely to be true functional variants. Thus far, only a few studies have addressed this in non-European populations, and our study is the first replication study in east Asians, with focus on both lipid trait and CAD associations. Replicating a study of candidate loci previously identified by GWA meta-analyses of European-descent populations, we have found some degree of ethnic diversity in lipid variants, while 18 of 22 tested loci are associated with lipid traits in the Japanese. The loci showing strong lipid associations were in good agreement between the ethnic groups except APOB, where associations with LDL-C and TG were relatively weak in the Japanese. Also, in the present study, we confirmed significant genetic impacts of 4 loci–SORT1, APOA5, LDLR, and APOE–on CAD in the Japanese. Of note, the effect size of APOE variants on CAD was significantly large in the Japanese. Moreover, it is of interest that as compared with the results for Europeans, the variance for LDL-C levels explained by individual SNP loci tended to be smaller in the Japanese, despite an overall cross-population consistency of genetic variants. For the most significant locus, APOE, 3 major isoforms are known to exist; E2 and E4 isoforms can be differentiated from E3 by rs7412 and rs429358, respectively. In agreement with a previous study of meta-analysis, E2 and E4 exerted GANT61 decreasing and increasing genetic effects on LDL-C, respectively, as compared to E3. While E2 carriers had a significantly decreased risk of CAD, E4 carriers showed no increased risk of CAD in the Japanese population, which is inconsistent with the previous reports. In addition, we found that effect sizes of APOE variants were heterogeneous between the current study and those previously reported. The genetic impacts on CAD seem to be more prominent for APOE rs7412 than the loci that we previously detected in the Japanese GWA scan. Because rs7412 and rs429358 themselves and their proxies are not included in the list of SNPs that are assayed by most of the GWA scan platforms, it is likely that these SNPs have failed to be tested for CAD associations in the previous GWA studies. As an approach to examining clinical relevance of lipidassociated SNPs, we assessed whether they are also associated with CAD in a manner consistent with established epidemiological relationships; i.e., SNP alleles that increase LDL-C or TG or that decrease HDL-C should be associated with increased risk of CAD, in proportion to the genetic effects on lipid traits. We inspected correlations between genetic effects on CAD risk and those on lipid traits.