Abstract Disclosure: N. Iwahashi: None. H. Umakoshi: None. T. Seki: None. C. Gomez-Sanchez: None. K. Mukai: None. M. Suematsu: None. Y. Umezawa: None. M. Oya: None. T. Kosaka: None. M. Seki: None. Y. Suzuki: None. Y. Horiuchi: None. K. Nishimoto: None. Y. Ogawa: None. Context: The adrenal cortex consists of zona glomerulosa (ZG), fasciculata (ZF), and reticularis. Aldosterone-producing cell clusters (APCCs) that express aldosterone synthase (CYP11B2) strongly can be frequently found in adult adrenals and harbor somatic mutations that are also present in aldosterone-producing adenomas (APAs). Primary aldosteronism is mainly caused by APAs or idiopathic hyperaldosteronism (IHA). We presume that APCCs are causing IHA and are precursors of APAs. However, the gene expression characteristics and especially the development of APCCs are not well understood. Objective: This study aimed to analyze the transcriptome of APCCs at single-cell resolution and infer the developmental trajectory. Methods: Single-cell RNA sequencing (scRNA-seq) was performed on two adult adrenals. Results: Immunohistochemical analyses confirmed the presence of APCCs in the two adrenals. scRNA-seq data of 6770 adrenal cells were obtained and 2928 high-quality cells were selected after quality control procedures. Unsupervised clustering and marker gene expressions such as CYP11A1 and NR5A1 identified 1765 adrenocortical cells, which were further divided into six clusters (B1-6). Using the list of APCC/ZG upregulated genes from our previously reported DNA microarray study, three clusters (B1, B3, and B6, totaling 923 cells) were identified as APCC/ZG cells. By further subclustering, the APCC/ZG cells were divided into 3 clusters (C1, C2, and C3). The APCC cluster (C3, 64 cells) and ZG cluster (C1, 447 cells) were identified. Cluster C2 seemed to be ZG-to-ZF transitional cells. RNA velocity analysis, which aimed to infer the direction of differentiation, inferred a direction of development from ZG cells to APCCs. Conclusion: Our results revealed the gene expression characteristics of APCC at single-cell resolution and show that some ZG cells remodel into APCCs. Presentation: Friday, June 16, 2023
The production of Bacillus subtilis extracellular proteases is under positive and negative regulation. The functional role of degR, one of the positive regulators, was studied in relation to the degS and degU gene products, which belong to the bacterial two-component regulatory system. Studies with a translational fusion between the Escherichia coli lacZ and the Bacillus subtilis subtilisin (aprE) genes indicated that the stimulatory site of DegR lay upstream of position -140, with the region upstream of position -200 being the major target. It was also found that degS and degU were epistatic to degR. These results suggested some relationship among the degR, degS, and degU gene products. The DegR protein was purified to homogeneity, and its in vitro effect on the phosphorylation reaction involving DegS and DegU was studied. For this purpose, a soluble-extract system in which the formation and dephosphorylation of DegU-phosphate could be examined was devised. The addition of DegR to the soluble-extract system enhanced the formation of DegU-phosphate. The enhancing effect was found to be due to the protection of DegU-phosphate from dephosphorylation. From these results, it was concluded that the positive effect of DegR on the production of the extracellular proteases is brought about by the stabilization of DegU-phosphate, which in turn may result in the stimulation of transcription of the exoprotease genes.
<p>PDF - 1735K, Table S1: Percentages of Fucci-HTC116 in S + G2 and G1 phases in mouse liver metastatic foci. Table S2: Numbers of MALDO-IMS measurement points in mouse liver metastatic foci. Figure S1: Frequency distribution of relative intensities of MALDI-IMS signal in mouse number 3. Figure S2: Synchronization Figure S3: Intracellular concentrations of metabolites Figure S4: Analysis of metabolites in HCT116 WT cells cultured under 1g/L glucose. Figure S5: Analysis of metabolites in HCT116 p53-null cells cultured under 3g/L glucose. Figure S6: Lactate efflux. Figure S7: Real-time RT-PCR analysis for monocarboxylate transporters (MCTs).</p>
Most c-type cytochromeshave a covalently bound heme linked by two thioether bonds to cysteine residues.Exceptions are the protozoan mitochondrial cytochromes c,1~ which have a heme c bound to the polypeptide chain through a single thioether bond.These cytochromes differ in their spectrophotometric properties, as exemplified by Euglena cytochrome c (c-558) .1)-4)Another mitochondrial c-type cytochrome, cytochrome c1, was discovered by Yakushiji and Okunuki5> and shows similar heme binding to typical cytochromes c.The cytochrome is recognized to be a component of cytochrome bc1 complex (ubiquinolcytochrome c oxidoreductase)where it donates electrons to cytochrome c in the respiratory chain.We suspected that Euglena cytochrome c1 might also have a heme bridged to its polypeptide chain through the unusual structure.In this paper we describe the isolation and spectral properties of Euglena cytochrome c1 and provide evidence for the presence of a single thioether bond binding the heme.Materials and methods.Euglena gracilis SM-ZK, a stable chloroplastlacking mutant, was used.Cells were cultured in Oda medium6~ containing 50 mg/l streptomycin, and kindly supplied by Toyo Jozo Co., Ltd.(Shizuoka, Japan) .Mitochondrial cytochrome bc1 complex was prepared by solubilization with Triton X-100 and chromatography on a DEAE-Toyopearl column (manuscript in preparation).Cytochrome c1 as a 32 kDa subunit was prepared as follows.The purified bc1 complex (320 nmol) was treated with 2-nitrophenylsulfenyl chloride in formic acid7> to remove the heme and the cysteine was carboxymethylated8)in the presence of 2-mercaptoethanol.After dialysis the sample was treated with sodium dodecylsulfate (SDS).For separation of cytochrome e1 from other subunits, gel filtration chromatography was performed on columns of Sephadex G-150 and Toyopearl HW-55F in the presence of SDS.For isolation of a peptide containing the heme-binding region of cytochrome c1, 50 nmol of the polypeptide was digested with trypsin and lysylendopeptidase.Peptides were separated by reversed phase high performance liquid chromatography and were analysed for amino acid composition.Amino acid sequence was determined using a gas-phase peptide sequences.Results and discussion.Purified Euglena cytochrome bc1 complex showed an atypical difference spectrum for cytochrome c1 compared to those of other complex (Fig. 1).The difference spectrum is of ascorbate-reduced minus ferricyanide-oxidized form of the ;purified complex.The a-band was at 561 nm and was slightly asymmetrical whereas cytochromes e1 from other sources exhibited a symmetricalx-peak at 552-553 nm.The presence of a subunit with covalently bound heme was indicated from the following results : (i) After acidic acetone-extraction of the cytochrome bc1
Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic β-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.
