Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca²⁺ concentration by uncaging of a caged-Ca²⁺ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.     

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1-7 after the injection, the proportion of the CD4+NK1.1+CD3(int) subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti-DNA antibodies. The proportion of CD1d(high)B cells in the liver was found to decrease on days 1-7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3(int) or NKT cells) but also some B cells (e.g. B-1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune-like state.     

Morphological characteristics of Schwann cells in the islets of Langerhans of the murine pancreas.

The present study demonstrated the three-dimensional architecture of peri-insular nerve plexuses in the murine pancreas by the combined use of light microscopy of S-100 immunostained sections, transmission electron microscopy (TEM) of thin sections, and scanning electron microscopy (SEM) of KOH digested tissues. By light microscopy of thin sections immunostained with anti-S-100 antibody, Schwann cells were often found on the margin of the islets as if delimiting the islet and exocrine parenchyma. In thick sections, Schwann cells of the islet connected their thin and slender processes with each other to form a delicate network on the surface of the islet. By TEM, Schwann cells were observed as an attenuated sheet that invested the surface of the islet. Axon terminals were usually found on the outer surface of these membranous Schwann cells. SEM of KOH digested tissues revealed that nerves reaching the islet spread on the insular surface. Schwann cells in this portion extended their thin membranous processes, which directly covered the basal part of several endocrine cells as a whole. Numerous axons with varicosities were usually found on the surface of these membranous Schwann cells, but sometimes crept beneath them. These findings indicate that "the interstitial cells" described by light microscopists are peculiar-shaped Schwann cells present in the islets. The functional significance of the rich innervation of the islets is also briefly discussed in the present study.     

Isolation and mapping of a polymorphic CA repeat sequence at the human VRK1 locus

VRK1 is a novel human putative serine/threonine kinase, and is located on chromosome 14 at band q32 where an autosomal recessive congenital microphthalmia (CMIC) is mapped. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human VRK1 gene. This polymorphism will be useful in genetic studies of disorders localized at the 14q32 region, such as CMIC. Received: October 8, 1998 / Accepted: October 16, 1998     

Functional interleukin 2 receptors on B cells lacking Tac antigens

Human B lymphoblastoid line, SKW 6-4, cells were induced to IgM-secreting cells by high concentrations of interleukin 2 (IL 2). These cells were found to be unreactive with anti-Tac antibody and did not express mRNA detectable for Tac antigen. In Scatchard plot analysis, low-affinity IL 2-binding sites were found on SKW 6-4 cells. Moreover, analysis of the IL 2-binding molecules revealed ones (molecular weight 70,000 and 75,000) distinct from Tac antigen. It is conceivable that IL 2 exerts its effect through its interaction with these novel IL 2-binding molecules in SKW 6-4 cells.     

Structure of the functional interleukin-2 receptor: Evidence for the association of human p55 and murine p75 molecules in a mouse T cell line

The structural basis of the high affinity interleukin-2 receptorwhich was previously reconstituted in a cultured murine T cellline, EL4 by expressing either wild-type Tac antigen complementaryDNA (cDNA) or a chimeric cDNA was characterized. The chimericcDNA encodes a membrane portion whose extracellular portionconsists of that of Tac antigen whereas transmembrane and cytoplasmicportions consists of those the human insulin beta chain. TheTac antigen/anti-Tac antibody complex was treated by chemicalcrosslinking reagents, purified by goat anti-mouse immunoglobulin(lg), and was analysed by SDS–PAGE. We here demonstrated the presence in mouse EL4 transfectantsof a novel membrane protein which is closely associated withthe products of transfected cDNAs in the absence of interleukln-2.The protein is 75 kDa in size and is detected in cells whichexpress high affinity interieukln-2 receptor but not in cellswhich only express low affinity interleukin-2 receptor. Thetransmembrane region and the cytoplasmic region of Tac antigenis not necessary for the formation of the complex consistingof Tac antigen and 75 kDa molecule, indicating that a murine75 kDa molecule associates with Tac antigen extra-cellularly.     

Evaluation of "M-point" in hepatic artery to identify left medial segment of liver. Angiographic study

The hepatic arteries of 122 patients were analysed on angiography to identify the left medial segment of the liver. Left medial arterial branches were classified into three types: type I arising from the left hepatic artery on the umbilical portion of the portal vein; type II arising from proximal portion of left hepatic artery before reaching the umbilical portion of the portal vein; type III arising from right hepatic artery. Incidence of each type is 37.2%, 35.8% and 27.0%, respectively. The artery frequently kinks at the right side of the umbilical portion of the portal vein and a total of incidence is 68% and that of each type is 23.5%, 89.8% and 100%, respectively. We call this characteristic kinking point of the left medial arterial branches, the "M-point".     

Impact of chronic kidney disease on oncological outcomes in patients with high-risk non-muscle-invasive bladder cancer who underwent adjuvant bacillus Calmette-Guérin therapy

ObjectivesTo investigate the impact of chronic kidney disease (CKD) on oncological outcomes in patients with high-risk non-muscle invasive bladder cancer (NMIBC) who underwent adjuvant induction bacillus Calmette-Guérin (BCG) therapy after transurethral resection of bladder tumor (TURBT).Materials and MethodsWe conducted a multi-institutional retrospective study assessing 209 patients with high-risk NMIBC who underwent TURBT and subsequent adjuvant induction BCG therapy from December 1998 to April 2019. Patients were divided into 2 groups: those with preoperative estimated glomerular filtration rate (eGFR) ≥ 60 ml/min/1.73 m² (non-CKD group), and those with eGFR < 60 ml/min/1.73 m² (CKD group). Primary endpoints were intravesical recurrence-free survival (RFS) and muscle-invasive bladder cancer (MIBC)-free survival. Background-adjusted multivariate analyses with the inverse probability of treatment weighting (IPTW) method using the propensity score were performed to evaluate the impact of CKD on intravesical RFS, MIBC-free survival, metastasis-free survival, cancer-specific survival, and overall survival. Moreover, multivariable analyses were performed to assess the impact of CKD on intravesical recurrence and MIBC progression, adjusting for the competing risk of death using the Fine-Gray competing risk regression model.ResultsMedian age and follow-up period after TURBT were 72 years and 45 months, respectively. Of 209 patients, 71 (34%) were diagnosed with CKD before TURBT. Background-adjusted multivariate analyses with the IPTW method indicated that CKD was significantly associated with shorter intravesical RFS, MIBC-free survival, metastasis-free survival, cancer-specific survival, and overall survival. In the Fine-Gray competing risk regression model, CKD showed significantly higher probabilities of intravesical recurrence and MIBC progression, with an adjusted subdistribution hazard ratio of 1.886 (95% confidence interval 1.069–3.330, P = 0.028) and 3.740 (95% confidence interval 1.060–13.20, P = 0.040), respectively.ConclusionsCKD presents a risk factor of poor oncological outcomes in patients with high-risk NMIBC who underwent adjuvant induction BCG therapy after TURBT.     

The effect of frailty on the quality of life and lower urinary symptoms following robot-assisted radical prostatectomy: A longitudinal analysis (FRARP-QL Study)

ObjectivesWe aimed to evaluate the effect of frailty on health-related quality-of-life (HRQOL) and lower urinary symptoms (LUTS) following robot-assisted radical prostatectomy (RARP) in patients with prostate cancer (CaP).Materials and MethodsWe longitudinally evaluated geriatric 8 (G8), HRQOL, and LUTS for 12 months in 118 patients with RARP from January 2017 to April 2020. Patients were divided into frail (G8 ≤14) and nonfrail (G8 >14) groups. We compared the effect of frailty on HRQOL and LUTS between the frail and nonfrail groups before and 12 months after RARP.ResultsThe median age of patients was 68 years. The number of patients in the frail and nonfrail groups were 41 and 77, respectively. No significant difference in patients’ background was observed between the groups, except for the presence of cardiovascular disease (22% vs. 7.8%, P = 0.041). There was no significant difference in HRQOLs and LUTS between the groups at baseline. Similarly, HRQOLs, LUTS, and pad-free continence rates were not significantly different between the groups at 12 months after RARP. In the nonfrail group, LUTS at 12 months following RARP significantly improved compared to those at the baseline, but it did not significantly improve in the frail group. Multivariable logistic regression analysis demonstrated that frailty was not significantly associated with LUTS worsening.ConclusionsFrailty was not significantly associated with the worsening of HRQOL, LUTS, and pad-free continence rates in patients treated with RARP.