Synergistic effect of cadmium chloride and acetaldehyde on cytotoxicity and its prevention by quercetin and glycyrrhizin

Cadmium chloride at concentrations of 10-50mM and acetaldehyde (AA) at 1-5mM showed synergistic toxic effects on V79 cells in vitro. Furthermore, synergistic effects of these chemicals were also observed in mutagenicities of the Hprt gene within certain dose ranges (cadmium chloride 5-10mM, and AA 1-2.5mM). Moreover, lipid peroxide formation, malondialdehyde (MDA) formation, detected by 2-thiobarbituric acid (TBA) reaction and the mitochondrial membrane potentials detected by rhodamine 123 uptake were significantly increased with the combined effect of cadmium and AA in V79. Thus, the cytotoxicity and genotoxicity displayed by combination of these chemicals can be considered to be associated with oxidative stress. Further, these effects were efficiently reduced by quercetin and less efficiently with glycyrrhizin.     

Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify amino acid residues essential for the thermostability of the SOD from Aquifex pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T(1/2) value of K12A was 210 min and that of the wild type was 175 min at 95 degrees C. However, the thermostability of the mutant E41A, which has a T(1/2) value of 25 min at 95 degrees C, was significantly decreased compared to the wild type of ApSOD. To explain the enhanced thermostability of K12A and thermolabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general significance of hydrogen bonds and ion-pair network in the thermostability of proteins.     

Identification of S-genotypes by PCR-RFLP in breeding lines of Brassica.

We developed a molecular method for the identification of the S-alleles of Brassicaceae, which belongs to the inbred line. This method is quicker and more precise than the existing methods. The genotype of the S-allele for 20 S-haplotypes of cabbage and 20 S-haplotypes of broccoli was determined by a pollination test. In order to identify the S-alleles, we performed PCR-RFLP with a mixture of the primers that are related to the S-locus glycoprotein (SLG) gene, which corresponds to the results of the pollination test. The selected primers amplified all of the single bands of about 1,150 bp in all 40 lines of cabbage and broccoli. Three out of 20 lines of cabbage were amplified by class I SLG specific primers, whereas all of the lines of the cabbage were amplified by class II SLG specific primers. Therefore, we could not classify class I and class II precisely by the class I and class II primers. However, 15 out of 20 lines of broccoli were amplified by the class I SLG specific primers. The remaining 5 lines were amplified with the class II SLG specific primers. We then digested the amplified PCR products with various restriction endonucleases and chose a restriction endonuclease, which accords exactly with the results of the diallel cross. The best one was HinfI. Its RFLP result was the same as that of the nucleotide sequence analysis. The 40 lines of cabbage and broccoli consisted of 16 different S-haplotypes. Therefore, the PCR-RFLP analysis was quicker and more precise in identifying the characteristics of S-haplotypes that are used in breeding. Also, we were able to check whether the lines could be mixed. The S-genotypes were difficult to determine due to the different flowering time.     

Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity

Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)₅R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P₂ over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.     

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing

Objectives: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.     

Computational modeling of epileptiform activities in medial temporal lobe epilepsy combined with <Emphasis Type="Italic">in vitro</Emphasis> experiments

Electrical stimulation of sub-cortical brain regions (the basal ganglia), known as deep brain stimulation (DBS), is an effective treatment for Parkinson’s disease (PD). Chronic high frequency (HF) DBS in the subthalamic nucleus (STN) or globus pallidus interna (GPi) reduces motor symptoms including bradykinesia and tremor in patients with PD, but the therapeutic mechanisms of DBS are not fully understood. We developed a biophysical network model comprising of the closed loop cortical-basal ganglia-thalamus circuit representing the healthy and parkinsonian rat brain. The network properties of the model were validated by comparing responses evoked in basal ganglia (BG) nuclei by cortical (CTX) stimulation to published experimental results. A key emergent property of the model was generation of low-frequency network oscillations. Consistent with their putative pathological role, low-frequency oscillations in model BG neurons were exaggerated in the parkinsonian state compared to the healthy condition. We used the model to quantify the effectiveness of STN DBS at different frequencies in suppressing low-frequency oscillatory activity in GPi. Frequencies less than 40 Hz were ineffective, low-frequency oscillatory power decreased gradually for frequencies between 50 Hz and 130 Hz, and saturated at frequencies higher than 150 Hz. HF STN DBS suppressed pathological oscillations in GPe/GPi both by exciting and inhibiting the firing in GPe/GPi neurons, and the number of GPe/GPi neurons influenced was greater for HF stimulation than low-frequency stimulation. Similar to the frequency dependent suppression of pathological oscillations, STN DBS also normalized the abnormal GPi spiking activity evoked by CTX stimulation in a frequency dependent fashion with HF being the most effective. Therefore, therapeutic HF STN DBS effectively suppresses pathological activity by influencing the activity of a greater proportion of neurons in the output nucleus of the BG.     

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.