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Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1. Graphical Abstract., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Although a close relationship between uraemic pruritus and serum calcium levels has been demonstrated for some time, the degree of pruritus was not always correlated with calcium concentrations. In the present study, we assessed calcium ion distribution in the skin of chronic haemodialysis patients with and without pruritus., Methods: We excluded patients with concomitant psoriasis or atopic dermatitis or with a previous history of allergy, those who had an arteriovenous fistula created prior to induction of haemodialysis, and patients with only mild pruritus. From the enrolled 22 haemodialysis patients, we obtained forearm skin samples during arteriovenous fistula surgery. These patients were divided into two groups based their grade of pruritus. The pruritus group included patients with moderate to severe grades of pruritus (n = 11, age 64 +/-13 years) and the non-pruritus group consisted of patients without pruritus (n = 11, age 59 +/-13 years). We compared the distribution of calcium ions in the epidermis between the two groups using the ion-capture method (oxalate-pyroantimonate-osmium technique). In addition, we examined and compared the groups for thicknesses of the basal, spinous and granular layers, as well as of the stratum corneum using an electron microscope., Results: The pruritus group had significantly higher calcium ion deposition in the extracellular fluid and cytoplasm of basal cells, and in the extracellular fluid, nuclei and cytoplasm of spinous cells compared with the non-pruritus group. In contrast, calcium ion depositions were similar between the two groups in the dermis/basal layer interface, the nucleus of basal cells, the nucleus and cytoplasm of granular cells, exterior of granular cells, the granular cells/stratum corneum interface, and in the interior and exterior of corneocytes. Although the stratum corneum was significantly thicker in the pruritus group than in the non-pruritus group, there were no differences in basal cell, spinal cell or granular cell layer thicknesses., Conclusion: In chronic haemodialysis patients with pruritus, the calcium ion concentration in the deepest layer of the epidermis was increased, which indicated a disrupted calcium ion gradient in the skin. These findings point to a role for increased skin calcium ion concentrations in the development and/or maintenance of uraemic pruritus. However, more extensive studies in larger patient cohorts will be necessary to confirm this hypothesis.

The release of arginine vasopressin (AVP) and atrial natriuretic hormone (ANH) and their involvement in renal water and electrolyte metabolism in primary aldosteronism in humans were studied. An oral acute water load (20 ml/kg body weight) was given to each of 12 patients before and after surgical removal of their aldosterone-producing adenoma(s). Plasma AVP and ANH were measured simultaneously, and renal water and electrolyte metabolism and tubular functions were determined. The same water load was given to seven normal subjects and the same parameters were determined. In the presence of mineralocorticoid excess before the operation, plasma AVP was relatively low compared with plasma osmolality (Posm), but was not suppressed in response to decreases in Posm after the water load. Baseline plasma ANH was high and increased further after the water load; urinary dilution and diuresis both remained normal. After the operation, baseline plasma AVP was normal and decreased in response to the decrease in Posm after the water load, with normal urinary dilution and diuresis. Baseline plasma ANH was normal, and did not increase after the water load. The ratio of urinary K and Na clearances and distal tubular reabsorption of Na increased before the operation. These results suggest that there are perturbations of AVP and ANH release in primary aldosteronism, despite the normal urinary dilution after a water load.

To examine the role of vasopressin (AVP) receptors in the regulation of the hemodynamics and release of atrial natriuretic peptide (ANP), and the participation of renal nerve inputs in the osmotic AVP release, hypertonic saline (HS) was infused into conscious, bilaterally nephrectomized rats with non-peptide, selective antagonists for the V1-receptor or V2-receptor of AVP. In the control group, HS alone increased mean arterial pressure, plasma ANP and AVP, plasma volume and plasma osmolality, and decreased the heart rate. In the V1-receptor antagonist group, an increase in the mean arterial pressure and a decrease in heart rate were completely abolished and an increase in plasma ANP was attenuated. In the V2-receptor antagonist group, increases in mean arterial pressure and plasma ANP and a decrease in heart rate were attenuated. However, the ratio of the changes in heart rate to the changes in mean arterial pressure in the V2-receptor antagonist group is significantly higher than that in the control group. In both experimental groups, increases in plasma AVP, plasma volume and plasma osmolality were not different from those in the control group. These results suggest that a HS-induced increase in mean arterial pressure is mediated by the pressor effect of AVP, mainly through V1-receptors, and that the depressor effect of AVP through V2-receptors may not influence tonically HS-induced hypertension. Moreover, HS-induced increase in plasma ANP is mediated mainly by increases in plasma volume and blood pressure, but may not be affected by a direct action of AVP to the heart. Renal afferent nerve inputs may not have effects on the regulation of osmotic AVP release.

A patient is reported who had undergone right adrenalectomy for pheochromocytoma and 15 yr later developed a recurrence in the same site complicated by the watery diarrhea, hypokalemia, achlorhydria syndrome. This tumor was histologically defined as a composite malignant pheochromocytoma-ganglioneuroblastoma (well differentiated type). Vasoactive intestinal polypeptide and catecholamine concentrations were elevated in both plasma and the tumor. The tumor somatostatin content also was high. The tumor was immunohistochemically determined to contain both vasoactive intestinal polypeptide and somatostatin.