Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface.

Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hß2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.

Cisplatin-induced programmed cell death ligand-2 expression is associated with metastasis ability in oral squamous cell carcinoma.

Programmed cell death ligands (PD-Ls) are expressed in tumor cells where they bind to programmed cell death-1, an immunocyte co-receptor, resulting in tumor cell evasion from the immune system. Chemotherapeutic drugs have been recently reported to induce the expression of PD-L, such as PD-L1, in some cancer cells. However, little is known regarding PD-L2 expression and its role in oral squamous cell carcinoma (OSCC). In this study, we examined the effect of cisplatin on the expression and regulation of PD-L2 in OSCC cell lines and analyzed malignant behavior in PD-L2-expressing cells using colony, transwell and transformation assays. In addition, we examined PD-L2 expression in the tumor tissues of OSCC patients using cytology and tissue microarray methods. In OSCC cell lines, cisplatin treatment upregulated PD-L2 expression, along with that of the drug efflux transporter ABCG2, via signal transducers and activator of transcription (STAT) 1/3 activation. Moreover, PD-L2-positive or PD-L2-overexpressing cells demonstrated upregulation in both invasion and transformation ability but not in proliferation compared with PD-L2-negative or PD-L2-silencing cells. PD-L2 expression was also observed in OSCC cells of cytology samples and tissue from OSCC patients. The intensity of PD-L2 expression was correlated with more malignant morphological features in the histological appearance and an invasive pattern. Our findings indicate that cisplatin-upregulated PD-L2 expression in OSCC via STAT1/3 activation and the expression of PD-L2 are likely to be associated with malignancy in OSCC. The PD-L2 expression in cisplatin-resistant OSCC cells may be a critical factor in prognosis of advanced OSCC patients.

Photothermal stress triggered by near-infrared-irradiated carbon nanotubes up-regulates osteogenesis and mineral deposition in tooth-extracted sockets.

PURPOSE: The bone regenerative healing process is often prolonged, with a high risk of infection particularly in elderly and diseased patients. A reduction in healing process time usually requires mechanical stress devices, chemical cues, or laser/thermal therapies. Although these approaches have been used extensively for the reduction of bone healing time, the exact mechanisms involved in thermal stress-induced bone regeneration remain unclear. METHODS: Photothermal stress (PTS) stimulation was carried out using a novel photothermal device, composed of an alginate gel (AG) including carbon nanotubes (CNT-AGs) and their irradiator with near-infrared (NIR) light. We investigated the effects of optimal hyperthermia on osteogenesis, its signalling pathway in vitro and mineral deposition in tooth-extracted sockets in vivo. RESULTS: The PTS (10 min at 42 °C, every day), triggered by NIR-induced CNT, increased the activity of alkaline phosphatase (ALP) in mouse osteoblast MC3T3-E1 cells in a time-dependent manner compared with the non-thermal stress control. PTS significantly induced the expression of osteogenic-related molecules such as ALP, RUNX2 and Osterix in a time-dependent manner with phosphorylated mitogen-activated protein kinases (MAPK). PTS increased the expression of heat shock factor (HSF) 2, but not HSF1, resulting in activation of heat shock protein 27. PTS significantly up-regulated mineral deposition in tooth-extracted sockets in normal and ovariectomised osteoporotic model mice in vivo. CONCLUSIONS: Our novel CNT-based PTS up-regulated osteogenesis via activation of heat shock-related molecules, resulting in promotion of mineral deposition in enhanced tooth-extracted sockets.

Reactive oxygen species promotes cellular senescence in normal human epidermal keratinocytes through epigenetic regulation of p16(INK4a.).

Reactive oxygen species (ROS) can cause severe damage to DNA, proteins and lipids in normal cells, contributing to carcinogenesis and various pathological conditions. While cellular senescence arrests the early phase of cell cycle without any detectable telomere loss or dysfunction. ROS is reported to contribute to induction of cellular senescence, as evidence by its premature onset upon treatment with antioxidants or inhibitors of cellular oxidant scavengers. Although cellular senescence is known to be implicated in tumor suppression, it remains unknown whether ROS initially contributed to be cellular senescence in normal human epidermal keratinocytes (NHEK) and their malignant counterparts. To clarify whether ROS induce cellular senescence in NHEKs, we examined the effect of hydrogen peroxide (H2O2) on the expression of cellular senescence-associated molecules in NHEKs, compared to in squamous carcinoma cells (SCCs). Hydrogen peroxide increased the number of cells positive in senescence associated-ß-galactosidase (SA-ß-Gal) activity in NHEKs, but not SCCs. The expression of cyclin-dependent kinase (CDK) inhibitors, especially p16(INK4a) was upregulated in NHEKs treated with H2O2. Interestingly, H2O2 suppressed the methylation of p16(INK4a), promoter region in NHEKs, but not in SCCs. Hydrogen peroxide also suppressed the expression of phosphorylated Rb and CDK4, resulting in arrest in G0/G1 phase in NHEKs, but not SCCs. Our results indicate that the ROS-induced cellular senescence in NHEKs was caused by the upregulation p16(INK4a) through demethylation in its promoter region, which is not detected in SCCs, suggesting that ROS-induced cellular senescence contributes to tumor suppression of NHEKs.

The potential role of transient receptor potential type A1 as a mechanoreceptor in human periodontal ligament cells.

Transient receptor potential type A1 (TRPA1) is reported to be a Ca(2+) -permeable channel and is activated by cold temperatures and mechanical stimuli in the hair cells and in dorsal root ganglion. Using a DNA microarray, we found that TRPA1 was significantly up-regulated in human periodontal ligament (hPDL) cells 2 d after intermittent mechanical stimulation (iMS) loading compared with unloaded cells. Although hPDL cells are known to respond to mechanical stimulation induced by occlusal force, little is known about the expression and functional role of TRPA1 in these cells. Therefore, we investigated the effects of iMS on TRPA1 expression and its signaling pathway in hPDL cells. Intermittent mechanical stimulation loading up-regulated TRPA1 expression in hPDL cells in a time-dependent manner, but had no effect on other mechanoreceptors. Furthermore, iMS significantly increased the phosphorylation of mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, and the expression of C-C chemokine ligand 2 (CCL2). Transient receptor potential type A1 agonists also increased MAPK phosphorylation and the intracellular Ca(2+) concentration. By contrast, inhibition or silencing of TRPA1 partially suppressed iMS-induced MAPK phosphorylation. In summary, iMS during occlusion activates TRPA1 and MAPK signaling in periodontal ligament tissues, suggesting that TRPA1 regulates the mechanosensitivity of occlusal force via activation of MAPKs in hPDL cells.

Characteristics of colonoscopic findings in the very elderly.

AIM: To determine the incidence and endoscopic types of colorectal lesions diagnosed with colonoscopy in elderly patients. METHODS: Consecutive Japanese patients who underwent colonic endoscopy between 1994 and 2007 (n = 5145; 2245 men and 2900 women, age 20-101 years) were examined retrospectively. Correlations between age, sex and number of lesions were analyzed. RESULTS: The incidence of advanced tumors was significantly correlated with increasing age in men (P = 0.02), and tumors were detected mainly in the sigmoid colon and rectum in both sexes. Right-sided colon cancer was significantly more common in women compared with men (P < 0.001). Polyps were detected throughout the colon, and their incidence was correlated significantly with increasing age in women (P = 0.01). Diverticula were frequently detected in the ascending and sigmoid colon in both sexes. Left-sided diverticula were significantly more common in women compared with men (P < 0.001). Lateral spreading tumors were detected mainly in the cecum in both sexes. Though the number of cases with angioectasia was small, angioectasia was slightly more common in the cecum and the ascending colon in women. CONCLUSIONS: In the present study, the incidence of advanced tumors correlated with increasing age in men, and right-sided cases were significantly more common in women than in men. The incidence of polyps correlated with increasing age in women. Left-sided diverticula were significantly more common in women than in men. Geriatr Gerontol Int 2016; 16: 1319-1323.

Undiagnosed cirrhosis occurs frequently in the elderly and requires periodic follow ups and medical treatments.

BACKGROUND: Autopsy examinations frequently reveal undiagnosed cirrhosis, but its characteristics have rarely been addressed in the elderly. METHODS: From 1597 consecutive autopsies, those of patients with liver cirrhosis were selected and their clinicopathological findings were examined. RESULTS: Seventy-six patients had liver cirrhosis; 18 of these patients (23.7%) were classified as an "undiagnosed" group and in that they had not been diagnosed as having cirrhosis before death. The remaining 58 patients were classified as a "clinical" group. Compared to the clinical group, the undiagnosed group demonstrated a significantly lower Child-Pugh score (7.1 +/- 1.9 vs 8.6 +/- 2.1; P < 0.01) and infrequent hepatocellular carcinoma (72.4% vs 5.6%; P < 0.0001). The undiagnosed group also demonstrated significantly lower complication rates of hepatic encephalopathy and esophageal varix, and a volume of ascites. The patients in the undiagnosed group were significantly older (79.9 +/- 8.1 vs 74.2 +/- 8.5 years; P < 0.01), and fewer patients died of liver-related causes (17% vs 67.2%; P < 0.0001). The etiology of cirrhosis was unknown in five patients in the undiagnosed group, and seven patients did not show any suggestive symptoms or imaging signs. CONCLUSION: Liver cirrhosis is often undiagnosed (23.7%) in the elderly. In the undiagnosed group, liver function was preserved and serious complications were infrequent. Because the diagnosis of cirrhosis leads to early identification of hepatocellular carcinoma and good prognosis, detailed examination and periodic follow ups should be performed when liver dysfunction is indicated, even in the elderly.

In vitro binding of hepatitis C virus to CD81-positive and -negative human cell lines.

AIM: The aim of the present study is to study the mechanism of entry of hepatitis C virus (HCV) into human cells. We examined the in vitro binding of HCV to various human cell lines with or without CD81 expression. METHODS: We first used three cell lines, two hepatocyte-derived, huH-7 and HepG2, and one colon cancer cell line, Cw2. Among them, HepG2 did not express TAPA-1 (CD81) on their surface but two others did. They were incubated with HCV + serum for 1 h and HCV-RNA in extracted RNA obtained from these cells was examined by using both a quantitative test and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We found that a significant amount of HCV-RNA was detected in huH-7 and HepG2 but not in Cw2. In addition, the titer of HCV-RNA in serum-incubated CD81-transfected HepG2 was similar to that of the non-transfected titer, and the binding between HCV and huH-7 was not inhibited by anti-CD81. Under the same condition, HCV-RNA tended to be detectable in serum-pulsed hepatocyte-derived cell lines, but not in the others. CONCLUSION: These results suggest that while CD81, as reported, specifically binds to HCV-E2 protein, the entry of HCV into human hepatocytes might be regulated by CD81-unrelated molecule(s).

A novel IgM class autoantibody to a hepatocyte-related 190 kDa molecule in patients with type 1 autoimmune hepatitis.

It has been reported that autoantibodies to hepatocytes are frequently found in patients with autoimmune hepatitis (AIH). To elucidate the nature of these hepatocyte-specific autoantibodies, we attempted to generate a hepatocyte-specific monoclonal antibody (MoAb) from Epstein-Barr virus-transformed peripheral blood mononuclear cells obtained from a patient with AIH. We established a single clone, 2E3, that continued to produce an immunoglobulin M (IgM) antibody (lambda-type). This MoAb had the following properties: it reacted mainly with hepatocyte-derived cell lines, rather than with other cell lines, and it reacted with liver tissue but not with other tissues. By immunoblot analysis, we found that this MoAb recognized a 190 kDa molecule on hepatocytes. The MoAb was able to kill hepatocyte-derived cell lines in the presence of fresh human serum. This cytotoxic effect was completely abrogated by heat inactivation of human serum prior to its addition to cell lines. In addition, an IgM autoantibody that recognized a 190 kDa molecule was also found in patients with AIH but not in those with chronic hepatitis C; its titer correlated significantly with serum alanine aminotransferase (ALT) levels in patients with AIH. In conclusion, we generated a human MoAb that recognizes a 190 kDa molecule on hepatocytes. Because of its ability to mediate complement-dependent cytotoxicity and the presence of similar IgM autoantibody in patients with AIH, we hypothesize this autoantibody may play a role in the immunopathogenesis of AIH.