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Pigs rarely develop cancer; however, tumour protein p53 (TP53)-modified pigs may have an increased incidence of cancer. In this study, two pigs with mosaic mutations induced by gene editing were compared to determine the role of the wild-type TP53 sequence in tumorigenesis and to speculate how amino acid changes in TP53 sequences are related to tumorigenesis. The pig without tumours had a wild-type TP53 sequence and a 1-bp deletion in the TP53 sequence that resulted in a premature stop codon. In contrast, the pig with nephroblastoma had 6- and 7-bp deletions in the TP53 sequence, resulting in the absence of two amino acids and a premature stop codon, respectively. Our results indicated that TP53 mutations with truncated amino acids may be related to tumour formation. [ABSTRACT FROM AUTHOR]

Aim: Non-alcoholic steatohepatitis (NASH) is a globally recognized liver disease. A methionine- and choline-deficient diet is used to induce NASH in mice; however, this diet also causes severe body weight loss. To resolve this issue, we examined the effects of methionine content in a high-fat and choline-deficient (HFCD) diet on body weight and the development of NASH in mice. Methods: C57BL/6J mice (male, 10 weeks of age) were fed an L-amino acid rodent (control) diet, high-fat (HF) diet, or HFCD diet containing various amounts of methionine (0.1–0.6% (w/w)) for 12 weeks. Plasma lipid levels, hepatic lipid content and inflammatory marker gene expression were measured, and a pathological analysis was conducted to evaluate NASH. Results: The 0.1% methionine in HFCD diet suppressed body weight gain, which was lower than that with control diet. On the other hand, the 0.2% methionine in HFCD diet yielded similar body weight gains as the control diet, while more than 0.4% methionine showed the same body weight gains as the HF diet. Liver weights and hepatic lipid contents were the greatest with 0.1% methionine and decreased in a methionine dose-dependent manner. Pathological analysis, NAFLD activity scores and gene expression levels in the liver revealed that 0.1% and 0.2% methionine for 12 weeks induced NASH, whereas 0.4% and 0.6% methionine attenuated the induction of NASH by HFCD diet. However, the 0.2% methionine in HFCD diet did not induce insulin resistance, despite the body weight gain. Conclusions: The 0.2% methionine in HFCD diet for 12 weeks was able to induce NASH without weight loss. [ABSTRACT FROM AUTHOR]

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins. [ABSTRACT FROM AUTHOR]

Potassium (K+)-uptake transport proteins present in prokaryote and eukaryote cells are categorized into two classes; Trk/Ktr/HKT, K+ channel, and Kdp belong to the same superfamily, whereas the remaining K+-uptake family, Kup/HAK/KT has no homology to the others, and neither its membrane topology nor crucial residues for K+ uptake have been identified. We examined the topology of Kup from Escherichia coli. Results from the reporter fusion and cysteine labeling assays support a model with 12 membrane-spanning domains. A model for proton-coupled K+ uptake mediated by Kup has been proposed. However, this study did not show any stimulation of Kup activity at low pH and any evidence of involvement of the three His in Kup-mediated K+ uptake. Moreover, replacement of all four cysteines of Kup with serine did not abolish K+ transport activity. To gain insight on crucial residues of Kup-mediated K+ uptake activity, we focused on acidic residues in the predicted external and transmembrane regions, and identified four residues in the membrane regions required for K+ uptake activity. This is different from no membrane-localized acidic residues essential for Trk/Ktr/HKTs, K+ channels and Kdp. Taken together, these results demonstrate that Kup belongs to a distinct type of K+ transport system. [ABSTRACT FROM AUTHOR]

Glycerol galactoside (GG; floridoside + isofloridoside) and porphyra-334 (P-334) are contained in nori (Susabinori Porphyra yezoensis and Asakusanori Porphyra tenera). Glycerol galactoside has been found to have bifidogenic growth stimulator activity and P-334 is known to have ultraviolet-absorbing activity in the UVA region of sunlight. These substances have, respectively, potential for application to pre-biotic foods and in cosmetics as a sunscreen. In the present study, to investigate the relationships between GG and P-334 contents and the quality of nori, we measured the GG and P-334 contents with other components (total protein, chlorophyll-a, β-carotene and phycobillins) that are related to the quality of nori samples produced from different production areas and with different qualities. We found that the GG content was closely negatively correlated with the contents of other components, whereas P-334 was positively correlated with the other components. From these results, it is suggested that low-quality nori is a potential source of GG, and as a source for P-334, scraps of nori produced during nori processing should be suitable. [ABSTRACT FROM AUTHOR]

Transmembrane α-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer. [ABSTRACT FROM AUTHOR]

Abstract: Multiple-site optical recording with a fast voltage-sensitive dye, absorption dye NK2761, was used to study the developmental organization of functional synaptic networks in the vagal pathway. Glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by vagus nerve stimulation was first detected from the nucleus of the tractus solitarius (NTS) at embryonic day 7 (E7) in chick embryos and E15 in rat embryos, when morphological differentiation of pre- and postsynaptic neurons is incomplete. When extracellular Mg2+ was removed, small EPSPs were elicited at E6 in chick embryos and E14 in rat embryos. These results suggest that synaptic function mediated by N-methyl-d-aspartate (NMDA) receptors is latently generated 1 day before the expression of glutamatergic EPSP. Functional synapses related to the glossophyaryngeal nerve appear to be generated at the same time as the vagus nerve, but their spatial distribution was different from that of the vagus nerve. We further investigated the development of second synaptic pathways from the NTS to higher centers, and found that neuronal circuits from the NTS are already generated when the primary afferents form functional synapses with NTS neurons. [Copyright &y& Elsevier]