Detection of dihydropyridine- and voltage-sensitive intracellular Ca2+ signals in normal human parathyroid cells

We recently showed dihydropyridine- and voltage-sensitive Ca²⁺ entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. To determine whether normal parathyroid cells have a similar extracellular Ca²⁺ entry system, cells were isolated from normal (non-hyperplastic) human parathyroid glands. Fluorescence signals related to the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_I) were examined in these cells. Cells loaded with fluo-3/AM showed a transient increase in fluorescence (Ca²⁺ transient) following a 10-s exposure to a 150 mM K⁺ solution in the presence of millimolar concentrations of external Ca²⁺. The Ca²⁺ transient was reduced by dihydropyridine antagonists or 0.5 mM Cd²⁺, but enhanced by FPL-64176, an L-type Ca²⁺-channel agonist. Ca²⁺ transients induced by the 10-s exposure to 3.0 mM extracellular Ca²⁺ ([Ca²⁺]_o) were also inhibited by dihydropyridine antagonists or 0.5 mM Cd²⁺. These results provide the first evidence that normal human parathyroid cells express a dihydropyridine-sensitive Ca²⁺ entry system that may be involved in the [Ca²⁺]_o-induced change in [Ca²⁺]_I. This system might provide a compensatory pathway for negative feedback regulation of parathyroid hormone secretion under physiological conditions.     

Serum α-fetoprotein level at treatment completion is a useful predictor of hepatocellular carcinoma occurrence more than one year after hepatitis C virus eradication by direct-acting antiviral treatment

Direct-acting antivirals (DAAs) have recently been developed to treat hepatitis C virus (HCV) infection, and interferon-free DAA treatment has improved liver function of HCV patients. The risk of hepatocellular carcinoma (HCC) occurrence following HCV eradication has been previously reported, but HCC may have been missed following imaging diagnosis before DAA administration in previous studies. Therefore, the present study aimed to identify definite predictors of HCC occurrence ≥1 year after DAA treatment. Among 956 patients receiving DAAs for HCV infection, 567 patients who achieved sustained virologic response with no history of HCC treatment were enrolled in this study between September 2014 and July 2021. The incidence of HCC in HCV-infected patients ≥1 year following DAA treatment, and the predictors contributing to HCC occurrence were identified using clinical characteristics and blood test results. In the present study, 25 patients developed HCC. The incidence of HCC was 1.4%, 3.2%, 4.9% and 6.8% at 2, 3, 4 and 5 years, respectively, from the end of treatment with DAAs. Multivariate logistic analysis revealed serum α-fetoprotein level at end of treatment (EOT-AFP) >3.8 ng/ml ≥1 year following treatment with DAAs (HR, 9.7; p < .0001) as an independent factor that may contribute to HCC occurrence following DAA treatment. In conclusion, serum EOT-AFP level may serve an important role in determining the risk of HCC occurrence ≥1 year after DAA treatment. Regular examinations are required even if serum EOT-AFP level is low at treatment completion.     

Biochemical,biological and structural properties of romidepsin (FK228) and its analogs as novel HDAC/PI3K dual inhibitors

Romidepsin (FK228, depsipeptide) is a potent histone deacetylase (HDAC) inhibitor that has FDA approval for the treatment of cutaneous and peripheral T‐cell lymphomas. We have previously reported that FK228 and its analogs have an additional activity as phosphatidylinositol 3‐kinase (PI3K) inhibitors, and are defined as HDAC/PI3K dual inhibitors. Because a combination of an HDAC inhibitor and a PI3K inhibitor induces apoptosis in human cancer cells in a synergistic manner, development of an HDAC/PI3K dual inhibitor will provide an attractive novel drug for cancer therapy. Using structure‐based optimization of the analogs, FK‐A11 was identified as the most potent analog. FK‐A11 inhibited phosphorylation of AKT and accelerated histone acetylation at lower concentrations, resulting in stronger cytotoxic effects than FK228 and the other analogs in human cancer cells. In this study, we have characterized the biochemical, biological and structural properties of FK228 analogs as PI3K inhibitors. First, FK‐A11 is an ATP competitive PI3K inhibitor. Second, FK‐A11 is a pan‐p110 isoform inhibitor. Third, FK‐A11 selectively inhibits PI3K among 22 common cellular kinases. Fourth, conformational changes of FK228 analogs by reduction of an internal disulfide bond have no effect on PI3K inhibitory activity, unlike HDAC inhibitory activity. Finally, molecular modeling of PI3K‐FK228 analogs and analyses of the binding affinities identified the structure that defines potency for PI3K inhibitory activity. These results prove our concept that a series of FK228 analogs are HDAC/PI3K dual inhibitors. These findings should help in the development of FK228 analogs as novel HDAC/PI3K dual inhibitors.     

Bactericidal Effects and Mechanism of Action of Olanexidine Gluconate,a New Antiseptic

Olanexidine gluconate [1-(3,4-dichlorobenzyl)-5-octylbiguanide gluconate] (development code OPB-2045G) is a new monobiguanide compound with bactericidal activity. In this study, we assessed its spectrum of bactericidal activity and mechanism of action. The minimal bactericidal concentrations of the compound for 30-, 60-, and 180-s exposures were determined with the microdilution method using a neutralizer against 320 bacterial strains from culture collections and clinical isolates. Based on the results, the estimated bactericidal olanexidine concentrations with 180-s exposures were 869 μg/ml for Gram-positive cocci (155 strains), 109 μg/ml for Gram-positive bacilli (29 strains), and 434 μg/ml for Gram-negative bacteria (136 strains). Olanexidine was active against a wide range of bacteria, especially Gram-positive cocci, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and had a spectrum of bactericidal activity comparable to that of commercial antiseptics, such as chlorhexidine and povidone-iodine. In vitro experiments exploring its mechanism of action indicated that olanexidine (i) interacts with the bacterial surface molecules, such as lipopolysaccharide and lipoteichoic acid, (ii) disrupts the cell membranes of liposomes, which are artificial bacterial membrane models, (iii) enhances the membrane permeability of Escherichia coli, (iv) disrupts the membrane integrity of S. aureus, and (v) denatures proteins at relatively high concentrations (≥160 μg/ml). These results indicate that olanexidine probably binds to the cell membrane, disrupts membrane integrity, and its bacteriostatic and bactericidal effects are caused by irreversible leakage of intracellular components. At relatively high concentrations, olanexidine aggregates cells by denaturing proteins. This mechanism differs slightly from that of a similar biguanide compound, chlorhexidine.     

Comparison of the clinical results of STA-MCA anastomosis and the medical treatment in the cerebral low perfusion patients with viable brain tissue

Abstract

The long-term clinical results of STA-MCA anastomosis as well as the medical treatments were compared in cases that were confined as having a focal cerebral perfusion deficit with viable brain tissue, based on either the drug induced EEG and evoked potential test (DEE test) and/or by positron emission tomography (PET). The criteria for viable cerebral tissue was determined by the following four conditions: (1) functional reversibility could be confirmed by the DEE test; (2) a haemodynamic process could be found in the DEE test; (3) a haemodynamic compromise could be confirmed in the PET study; (4) misery perfusion could be confirmed in the PET study.

From 1975 to 1989, 55 cases were confirmed as having viable brain tissue according to the DEE test and the PET study. Of the 55 casesbypass surgery was performed on 35. Conservative treatment was given to the other 20 cases. There were 3 cases of perioperative neurological deterioration. One was permanent and the other 2 were transcient. Results of the long-term follow up are as follows. Ipsilateral attack: 1 case (2.0%) received surgery, and 7 cases (35%) received conservative treatment. Re-attack in the contralateral or posterior circulation: 6 cases (17.2%) received surgery, and 1 case (5%) received conservative treatment. Seventy-seven per cent of the surgical cases improved or had no change in the final functional statuswhile only 55% of the conservative group either improved or showed no change. The incidence of ipsilateral cerebral ischaemia was significantly low in the surgical group. Contralateral and/or posterior circulation ischaemia tended to be high in this group, however. The final functional state was also better in the surgical group. These results show the usefulness of STA-MCA anastomosis in cases that are confirmed to have viable cerebral tissue by either the DEE test or the PET.