Nogo-B Promotes Endoplasmic Reticulum Stress-Mediated Autophagy in Endothelial Cells of Diabetic Nephropathy.

Aims: Endothelial cells are the critical targets of injury in diabetic nephropathy (DN), and endothelial cell lesions contribute to the disease progression. Neurite outgrowth inhibitor B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, plays a pivotal role in vascular remodeling after injury, and maintains the structure and function of the ER. Yet, the role of Nogo-B in the regulation of ER stress and endothelial cell injury remains largely unknown. Herein, we tested the hypothesis that Nogo-B activates ER stress-mediated autophagy and protects endothelial cells in DN. Results: The level of Nogo-B was decreased in glomerular endothelial cells in biopsy specimens from DN patients. In vivo and in vitro studies have shown that silencing Nogo-B activated ER stress signaling, and affected the expression of autophagy-related marker early growth response 1 and microtubule-associated protein light chain 3 (LC3) in endothelial cells in hyperglycemic condition. Conclusion and Innovation: These results denote that Nogo-B contributes to ER stress-mediated autophagy and protects endothelial cells in DN, providing new evidence for understanding the role of ER stress-mediated autophagy in endothelial cells of DN.

Application Effect of Multi-Dimension Nursing Combined with GRACE Scoring System in Patients with Atrial Fibrillation after Green Precision Catheter Radiofrequency Ablation.

OBJECTIVE: To explore the application effect of multi-dimensional nursing combined with the Global Registry of Acute Events (GRACE) scoring system in the nursing of patients with atrial fibrillation after radiofrequency ablation with green precision catheter radiofrequency ablation. METHODS: A total of 274 patients diagnosed with atrial fibrillation undergoing green precision catheter radiofrequency ablation were collected from the Department of Cardiology at our hospital in a retrospective study. After the inclusion, exclusion, diagnostic criteria and physical examination, all the subjects underwent green precision catheter radiofrequency ablation. According to various nursing methods that were adopted, they were divided into two groups with 7-14 days of nursing intervention by digital randomization: the study group (multi-dimensional nursing combined with GRACE scoring system evaluation, n = 136 cases) and the control group (postoperative routine nursing, n = 138 cases). The MOS item short from health survey (SF-36) score, Hamilton anxiety scale (HAMA) score, Hamilton depression scale (HAMD) score, complication rates and the nursing quality of the two groups were observed. RESULTS: After multi-dimensional nursing combined with the GRACE in-hospital scoring system for stratified nursing in the study group, SF-36 scores in both groups increased after conventional nursing in the control group, but there was a statistical difference between the study group and the control group (p < 0.05). HAMA score and HAMD score decreased, and there were statistical differences between the study group and the control group (p < 0.05). The comparison between the study group and the control group showed that "Cardiac tamponade", "Atrioventricular block", "Peripheral vascular injury" and the total incidence of complications were statistically different (p < 0.05). The basic satisfaction, number of satisfaction and total satisfaction rate of the study group were higher than those of the control group, and the difference was statistically significant (p < 0.05). CONCLUSIONS: Multi-dimensional nursing combined with the GRACE scoring system in the nursing care of patients with atrial fibrillation after radiofrequency ablation with the green precision catheter, improves the quality of life, alleviates negative emotions, reduces the incidence of complications, and results in better quality of nursing care.

Application Effects of NNN-link Care Model in Patients with Coronary Heart Disease.

OBJECTIVE: To investigate the effect of a NNN-linked care model applied in elderly patients with coronary heart disease. METHODS: A total of 120 elderly patients with coronary heart disease admitted to the hospital from January, 2023 to May, 2023 were randomly divided into two groups of 60 cases respectively. The control group received routine intervention, and the observation group received the NNN-linked care model. Changes in cardiac function, the ability for self-care, and quality of life were recorded between the groups before and after the intervention. RESULTS: Indices of cardiac function in the observation group were higher than those of the control group after 3 weeks (p < 0.05). Compared with the control group, the total score for the ability for self-care and the scores of each dimension of the observation group were higher after 3 weeks of intervention (p < 0.05). The scores of quality of life of the observation group were higher in comparison with the control group after 3 weeks of intervention (p < 0.05). CONCLUSION: The application of the NNN-linked care model to elderly patients with coronary heart disease can improve the ability for self-care, increase cardiac function and improve the quality of life.

"Guide" of muscone modification enhanced brain-targeting efficacy and anti-glioma effect of lactoferrin modified DTX liposomes.

Glioma is one of the most aggressive malignant diseases for human health. It is difficult to resect completely due to their invasiveness. The targeted delivery, as a noninvasive approach, is a major strategy for the development of treatments for brain tumors. Lactoferrin (Lf) receptors are over-expressed in both brain endothelial cells and glioma cells. Macromolecular Lf modified nanoparticles have been shown to enhance the brain targeting. Muscone is a "guide" drug that have been demonstrated to promote liposomes into the brain by modification. To further enhance the brain-targeting efficacy of Lf modified carriers, we designed that Lf and muscone dual-modified liposomes cross blood-brain barrier (BBB) and target to brain for enhanced docetaxel (DTX) brain delivery. The results showed that we successfully prepared Lf and muscone dual-modified liposomes (Lf-LP-Mu-DTX), the number of Lf molecules connected to the surface of per liposome was 28. Lf-LP-Mu-DTX increased uptake in both U87-MG cells and hCMEC/D3 cells, enhanced penetration of U87-MG tumor spheroid and in vitro BBB model, had better in vitro and in vivo anti-tumor effects. In conclusion, "guide" of muscone modification enhanced brain-targeting efficacy of Lf modified liposomes, Lf and muscone dual-modified docetaxel loaded liposomes present a potential brain-targeting drug delivery system for use in the future treatment of gliomas.

Association of Dietary Niacin Intake with Diabetes in Adults in the United States.

OBJECTIVE: Previous studies have shown inconsistent associations between niacin supplementation and diabetes, and little is known about the relationship between dietary niacin intake and the risk of diabetes in the general population. Our study aimed to explore the association between dietary niacin intake and the risk of diabetes in the adult population in the United States. METHODS: Data from the 2005-2016 National Health and Nutrition Examination Surveys were analyzed. Diabetes was diagnosed according to the American Diabetes Association criteria. Multivariate logistic regression models were used to estimate the association between dietary niacin intake and diabetes. Covariates included age, sex, race, family income, educational level, drinking status, smoking status, marital status, and physical activity. RESULTS: This study included 24494 participants, of which 13.63% had diabetes. In the fully adjusted model, a high niacin intake was significantly associated with a reduced risk of diabetes in a dose-dependent manner. When extreme quintiles of niacin intake were compared, the multivariable-adjusted odds ratio was 0.66 (95% confidence interval: 0.49, 0.88) for diabetes, and per ten-unit increment in dietary niacin intake was associated with a 14% lower risk of diabetes. When niacin intake was less than 15.01 mg/d, a ten-unit increment in niacin intake was associated with a 24% higher risk of diabetes. However, the effect was not statistically significant. CONCLUSIONS: Our results suggest that the consumption of adequate amounts of niacin can reduce the risk of diabetes. Furthermore, this protective effect disappeared when the niacin intake was insufficient (less than 15.01 mg/d).

Cigarette Smoking Increases the Risk of Type 2 Diabetes Mellitus in Patients with Non-Alcoholic Fatty Liver Disease: A Population-Based Cohort Study.

OBJECTIVE: Cigarette smoke and non-alcoholic fatty liver disease are risk factors for type 2 diabetes mellitus. However, the impact of smoking on diabetes risk among patients with non-alcoholic fatty liver disease remains unclear. METHODS: This study included 15,464 Japanese individuals. We defined non-alcoholic fatty liver disease based on abdominal ultrasound findings where excess alcohol intake and other liver diseases were excluded. We used Cox proportional regression analysis to identify risk factors for type 2 diabetes onset. RESULTS: During 16,446 person-years of follow-up, 223 of 2,714 non-alcoholic fatty liver disease patients developed type 2 diabetes; the cumulative incidence rate of type 2 diabetes was 13.6 per 1,000 person-years. The proportions of never, former, and current smokers (self-report) were 35.3%, 31.1%, and 33.6%, and 88.5%, 5.1%, and 6.4% in men and women, respectively. In a Cox regression model adjusted for sex, age, body mass index, waist circumference, alcohol intake, exercise, and alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, lipid profiles, and blood pressure values, relative to never smokers, current smokers with non-alcoholic fatty liver disease had an increased risk of type 2 diabetes (hazard ratio=2.05; 95% confidence interval: 1.43-2.94). In addition, former smoking affected the risk of type 2 diabetes; however, this effect was not statistically significant. CONCLUSIONS: This longitudinal study showed that current smoking may act as a "second hit" and increase the risk of type 2 diabetes in patients with non-alcoholic fatty liver disease.

Combined integrin αvß3 and lactoferrin receptor targeted docetaxel liposomes enhance the brain targeting effect and anti-glioma effect.

The integrin αvß3 receptor and Lactoferrin receptor (LfR) are over-expressed in both cerebral microvascular endothelial cells and glioma cells. RGD tripeptide and Lf can specifically bind with integrin αvß3 receptor and LfR, respectively. In our study, RGD and Lf dual-modified liposomes loaded with docetaxel (DTX) were designed to enhance the brain targeting effect and treatment of glioma. Our in vitro studies have shown that RGD-Lf-LP can significantly enhance the cellular uptake of U87 MG cells and human cerebral microvascular endothelial cells (hCMEC/D3) when compared to RGD modified liposomes (RGD-LP) and Lf modified liposomes (Lf-LP). Free RGD and Lf competitively reduced the cellular uptake of RGD-Lf-LP, in particular, free RGD played a main inhibitory effect on cellular uptake of RGD-Lf-LP in U87 MG cells, yet free Lf played a main inhibitory effect on cellular uptake of RGD-Lf-LP in hCMEC/D3 cells. RGD-Lf-LP can also significantly increase penetration of U87 MG tumor spheroids, and RGD modification plays a dominating role on promoting the penetration of U87 MG tumor spheroids. The results of in vitro BBB model were shown that RGD-Lf-LP-C6 obviously increased the transport of hCMEC/D3 cell monolayers, and Lf modification plays a dominating role on increasing the transport of hCMEC/D3 cell monolayers. In vivo imaging proved that RGD-Lf-LP shows stronger targeting effects for brain orthotopic gliomas than that of RGD-LP and Lf-LP. The result of tissue distribution confirmed that RGD-LF-LP-DTX could significantly increase brain targeting after intravenous injection. Furthermore, RGD-LF-LP-DTX (a dose of 5 mg kg-1 DTX) could significantly prolong the survival time of orthotopic glioma-bearing mice. In summary, RGD and LF dual modification are good combination for brain targeting delivery, RGD-Lf-LP-DTX could enhance brain targeting effects, and is thus a promising chemotherapeutic drug delivery system for treatment of glioma.

Muscone/RI7217 co-modified upward messenger DTX liposomes enhanced permeability of blood-brain barrier and targeting glioma.

Rationale: The dual-targeted drug delivery system was designed for enhancing permeation of the blood-brain barrier (BBB) and providing an anti-glioma effect. As transferrin receptor (TfR) is over-expressed by the brain capillary endothelial (hCMEC/D3) and glioma cells, a mouse monoclonal antibody, RI7217, with high affinity and selectivity for TfR, was used to study the brain targeted drug delivery system. Muscone, an ingredient of traditional Chinese medicine (TCM) musk, was used as the "guide" drug to probe the permeability of the BBB for drug delivery into the cerebrospinal fluid. This study investigated the combined effects of TCM aromatic resuscitation and modern receptor-targeted technology by the use of muscone/RI7217 co-modified docetaxel (DTX) liposomes for enhanced drug delivery to the brain for anti-glioma effect. Methods: Cellular drug uptake from the formulations was determined using fluorescence microscopy and flow cytometry. The drug penetrating ability into tumor spheroids were visualized using confocal laser scanning microscopy (CLSM). In vivo glioma-targeting ability of formulations was evaluated using whole-body fluorescent imaging system. The survival curve study was performed to evaluate the anti-glioma effect of the formulations. Results: The results showed that muscone and RI7217 co-modified DTX liposomes enhanced uptake into both hCMEC/D3 and U87-MG cells, increased penetration to the deep region of U87-MG tumor spheroids, improved brain targeting in vivo and prolonged survival time of nude mice bearing tumor. Conclusion: Muscone and RI7217 co-modified DTX liposomes were found to show improved brain targeting and enhanced the efficacy of anti-glioma drug treatment in vivo.

Resveratrol attenuates pulmonary embolism associated cardiac injury by suppressing activation of the inflammasome via the MALAT1­miR­22­3p signaling pathway.

Resveratrol (RS) has been reported to prevent the development of cardiac injury induced by pulmonary embolism (PE). The present study aimed to explore the potential mechanism of RS involved in cardiac injury induced by PE. A luciferase assay was conducted to detect the effect of RS on promoter efficiency of metastasis associated lung adenocarcinoma transcript 1 (MALAT1), in­silico analysis and luciferase assays were performed to explore the regulatory relationship between MALAT1, microRNA (miR)­22­3p and NLRP3. Reverse transcription PCR, western blot analysis and ELISA were carried out to examine MALAT1, miR­22­3p, NLRP3, ASC, Caspase­1, interleukin (IL)­1ß and IL­18 among different animal model groups, including the sham group, PE associated cardiac injury group and PE associated cardiac injury plus RS group. The results revealed that RS downregulated promoter efficiency of MALAT1 and MALAT1 directly targeted miR­22­3p, and luciferase activity of MALAT1 was inhibited by miR­22­3p, and furthermore miR­22­3p inhibited the expression of NLRP3 by binding to complementary sequences in the 3' untranslated region of NLRP3. MALAT1, NLRP3, ASC, Caspase­1, IL­1ß and IL­18 levels were much increased, while miR­22­3p level was much decreased in PE associated cardiac injury group compared with the sham group, while the RS upon the PE associated cardiac injury group slightly reduced the upregulated MALAT1/NLRP3 level and elevated the downregulated miR­22­3p level. In conclusion, it was demonstrated that RS has been demonstrated to prevent the development of cardiac injury induced by PE via modulating the expression of MALAT1 and further affect miR­22­3p and NLRP3.

How great is current curative expenditure and catastrophic health expenditure among patients with cancer in China? A research based on "System of Health Account 2011".

In recent years, the incidence and mortality of cancer have witnessed a dramatic increase. Cancer has already caused severe economic burdens on society, especially in developing countries and has become a major public health concern. This study evaluates the medical economic burden, including total current curative expenditure (CCE) and catastrophic health expenditure (CHE) on cancer in Liaoning Province, China. A total of 252 medical institutions were investigated with multistage stratified cluster random sampling. We established a standardized database of 3 532 517 samples. "System of Health Account 2011", a new internationally recognized accounting system, was established to analyze the CCE on six most common cancers. CHE were estimated from the extracted 1344 patients with cancer, which performed a cross-sectional study. The association of individual and contextual factors with CHE was evaluated using logistic regression models. CCE for all the patients with the six types of cancer was 2801.38 million CNY in Liaoning Province, the highest of which was lung cancer. The incidence of CHE was 42.78%, while the threshold was 40%. The average and relative distance were 10.41% and 24.32%, respectively. Influencing factors were length of stay, type of health insurance, location of household, etc. Our findings highlight the need to address medical economic burden in the cancer population. Households with the cancer are more likely to incur CHE. Financial intervention to prevent it should target on poor households. We provide suggestions in aspects of health insurance and health service management to reduce CHE.

Activation of dendritic cells by low molecular weight oyster polysaccharides.

Dendritic cells play a primary role in antigen presentation to CD4+ T cells, which initiate acquired immune responses. Therefore, determining positive modulators of dendritic cell activation to improve therapeutic approaches for cancer treatment might be useful. We here investigated the effects of low molecular weight oyster polysaccharides (LMW-OPS) on bone marrow-derived dendritic cells (BMDCs) obtained from mice. LMW-OPS increased the surface expression of major histocompatibility complex class II (MHC-II), CD40 and CD86 in BMDCs and induced the secretion of tumour necrosis factor (TNF)-α and interleukin (IL)-12, which were significantly decreased in the BMDCs derived from MyD88-/- mice but not from the lipopolysaccharide-resistant C3H/HeJ mice. BMDCs treated with LMW-OPS augmented allogeneic CD4+ T cell expansion and enhanced secretion of IL-2 and interferon (IFN)-γ but not IL-4. LMW-OPS induced significant increases in ERK and p38 MAPK phosphorylation, but not c-Jun N-terminal kinase (JNK) phosphorylation, in BMDCs. Our results indicate that, in part, LMW-OPS can induce maturation of BMDCs in a MyD88-dependent and Toll-like receptor (TLR) 4-independent manner. LMW-OPS may enhance acquired immunity by modulating the function of dendritic cells.

Opposing roles for Smad2 and Smad3 in peritoneal fibrosis in vivo and in vitro.

Peritoneal fibrosis is a major cause of ultrafiltration failure in patients receiving continuous ambulatory peritoneal dialysis. Transforming growth factor (TGF)-ß1 is an important mediator in this process; however, its signaling mechanisms had not been explored. Thus, we examined TGF-ß1/Smad signaling in human peritoneal biopsy specimens associated with continuous ambulatory peritoneal dialysis. We found that TGF-ß/Smad2/3 signaling was highly activated in patients with increased collagen deposition and thickening of the peritoneal membrane who were receiving continuous ambulatory peritoneal dialysis. Long-term exposure of wild-type mice to 4.25% peritoneal dialysis solution for 30 days induced significant peritoneal fibrosis with impaired peritoneal equilibrium, which was prevented in Smad3 knockout mice. In contrast, conditional Smad2 gene deletion in the peritoneum exacerbated peritoneal fibrosis and dysfunction. The contrasting roles of Smad2 and Smad3 in peritoneal fibrosis were also examined in vitro. Cultured mesothelial cells from Smad3 knockout mice were resistant to TGF-ß1-induced collagen I production and the transition toward a myofibroblast phenotype as seen in wild-type cells, whereas Smad2 deficiency in mesothelial cells failed to modulate the profibrotic response to TGF-ß1. In conclusion, this study found activation of TGF-ß/Smad signaling in peritoneal fibrosis in patients receiving continuous ambulatory peritoneal dialysis and identifies opposing roles for Smad2 and Smad3 in peritoneal dialysis-associated peritoneal fibrosis. These findings provide a mechanistic basis for future therapies targeting TGF-ß/Smad signaling in peritoneal fibrosis.

Preparative separation of polyphenols from the flowers of Paeonia lactiflora Pall. by high-speed counter-current chromatography.

High-speed counter-current chromatography was used to separate and purify polyphenols for the first time from the flowers of Paeonia lactiflora Pall. with two solvent systems as follows: petroleum ether-ethyl acetate-H2O (1:9:10, v/v/v/v) and petroleum ether-ethyl acetate-butanol-H2O (1:9:0.5:10, v/v/v/v). Eight compounds were separated successfully in single run which were identified as quercetin-3-O-(6″-O-galloyl)-glucoside (I, 41.5 mg), 1,2,3,4,6-trigalloyl-ß-D-glucose (II, 106.2mg), quercetin-3-O-ß-D-glucoside (III, 42.3 mg), kaempferol-3-O-(6″-O-galloyl)-glucoside (IV, 23.5 mg), isohamnetin-3-O-ß-D-glucoside (V, 34.1 mg), kaempferol (VI, 14.8 mg), kaempferol-3-O-ß-D-glucoside (VII, 32.6 mg), kaempferol-7-O-ß-D-glucoside (VIII, 23.8 mg) by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The purities of compounds I-VIII were all over 97.0% as determined by HPLC.

Vandetanib-induced inhibition of neuroblastoma cell migration and invasion is associated with downregulation of the SDF-1/CXCR4 axis and matrix metalloproteinase 14.

Rearranged during transfection (RET) is widely expressed in neuroblastoma (NB) and partly contributes to high metastatic potential and survival of NB. The aim of the present study was to investigate whether vandetanib (a RET inhibitor) inhibits proliferation, migration and invasion of NB cells in vitro. The effects of vandetanib on the proliferation, apoptosis and cell cycle and on RET phosphorylation of SK-N-SH and SH-SY5Y cells were evaluated in vitro. The migration and invasion potential of vandetanib-treated NB cells were analyzed using Transwell cell migration and invasion assays, respectively. qPCR, western blotting and immunofluorescence were used to detect mRNA and protein levels in NB cells treated with vandetanib. Our data demonstrated that vandetanib inhibits the proliferation of SK-N-SH and SH-SY5Y cells and that this inhibition is mediated by the induction of G1 phase cell cycle arrest at lower concentrations and by apoptosis at higher concentrations. In the presence of vandetanib, the migration and invasion of two NB cell lines were markedly decreased compared with the control group (p<0.01). In addition, our data showed that the levels of C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinase 14 (MMP14) mRNA expression in NB cell lines treated with vandetanib were significantly lower than those in the cells that were treated with vehicle (p<0.01) and similar results were obtained for protein levels as determined by western blotting and immunofluorescence analysis. Vandetanib may inhibit the proliferation, migration and invasion of NB cells in vitro. The potential mechanisms for the inhibition of NB migration and invasion by vandetanib may partly be attributed to the ability of vandetanib to suppress the expression of CXCR4 and MMP14 in human NB cells.

Expression of the novel all-trans retinoic acid-related resistance gene HA117 in pediatric solid tumors.

This study aimed to detect the protein expression of HA117 in pediatric solid tumors. Immunohistochemistry was performed to detect the expression of HA117 and P-gp in pediatric solid tumors. In Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), nephroblastoma (WT), and neuroblastoma (NB), the positive expression rate of HA117 was 65.4%, 58.3%, 81.3%, and 74.1%, and that of P-gp was 57.7%, 70.8%, 65.6%, and 66.7%, respectively. HA117 expression was closely related to the clinical stage of HL (P=0.004) and to the International Prognostic Index score, mediastinal lesions, and clinical stages of NHL (P=0.01, 0.03, and 0.01). The expression of HA117 in WT was higher than in adjacent normal tissues, but there was no statistical significance (P=0.21). The positive expression of HA117 in NB was markedly higher than that in normal tissues (P=0.002), which closely associated with histologic type and lymph node metastasis (P=0.03 and 0.001). Spearman correlation analysis revealed that HA117 expression was not correlated with P-gp in these 4 tumors. This suggests that HA117 might be an important resistance gene in pediatric solid tumors. The mechanism underlying the resistance to all-trans retinoic acid conferred by HA117 is different from that of P-gp.

Expression patterns of CXCR4 in different colon tissue segments of patients with Hirschsprung's disease.

C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in a wide range of physiological and pathological processes, including the migration of stem cells, such as neural crest-derived cells. Hirschsprung's disease (HSCR), a developmental disorder characterized by the absence of ganglion cells, is regarded as the consequence of the premature arrest of the craniocaudal migration of neural crest-derived cells (NCDCs) in the gastrointestinal tract during the development of the enteric nervous system (ENS). In this study, colon tissue samples from 61 HSCR patients were surgically collected and divided into aganglionic, oligoganglionic and normal ganglionic segments. Quantitative real-time polymerase chain reactions (PCR), Western blotting, and immunohistochemical and immunofluorescence staining were performed to analyze the expression levels and patterns of CXCR4 in different colon tissue segments. The expression levels of CXCR4 mRNA and protein in the aganglionic segments were decreased compared to the normal ganglionic and oligoganglionic colon segments (p<0.01). Immunohistochemical staining showed that intensive CXCR4 staining was detected in the ganglion cells and the supporting glial cells in the ganglion in control colon specimens and normal ganglionic and oligoganglionic colon segments from the HSCR patients; however, CXCR4 staining was significantly decreased in the aganglionic colon segments. Immunofluorescence staining showed that CXCR4 staining was mainly detected in the ganglia where RET-positive ganglion cells were observed. Elucidating CXCR4 expression patterns in colon segments could be the basis for further investigations of the potential role of CXCR4 in ENS development.

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase.

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

Expression of BMP2, TLR3, TLR4 and COX2 in colorectal polyps, adenoma and adenocarcinoma.

The initiation and development of colorectal cancer is closely associated with the malignant transformation of colorectal polyps. The aim of this study was to analyze the expression of the bone morphogenetic protein-2 (BMP2), toll-like receptor 3 (TLR3), TLR4 and cyclooxygenase-2 (COX2) proteins in colorectal polyps, adenoma and adenocarcinoma. An immunohistochemical streptavidin-peroxidase (SP) method was used to examine the expression of MBP2, TLR4, TLR3 and COX2 in 20 colorectal juvenile polyps and 15 colorectal polyps of hamartomatous polyposis obtained from children, and 20 colorectal adenomas and 20 colorectal adenocarcinomas obtained from adults. A comparison of the expression levels of TLR3 among the groups revealed a gradual downward trend from the colorectal juvenile polyp group to the colorectal hamartomatous polyposis, adenoma and adenocarcinoma groups, respectively. The expression level of TLR3 was significantly lower in the colorectal adenocarcinoma group (p<0.05). The expression levels of TLR3, TLR4 and BMP2 were significantly different among the colorectal juvenile polyp, hamartomatous polyposis, adenoma and adenocarcinoma groups. These three protein molecules may be significant in the development and malignant transformation of colorectal polyps.

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts.

The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1ß, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/ß, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

[Urine protein from patients with lupus nephritis stimulates transdifferentiation and high expression of collagen in renal tubular cells].

OBJECTIVE: To investigate the effects of urine protein on the renal tubular-interstitial fibrosis in the patients with lupus nephritis (LN). METHODS: Protein was isolated and purified from the urine of six patients with primary LN, 1 male and 5 females, aged 27.4, and incubated with renal tubular cells of the line HK-2 for 0, 1, 2, 12, 24, or 48 h respectively. The mRNA expressions of transforming growth factor beta1(TGF-beta1), collagen I (COL I), and alpha-smooth muscle actin (alpha-SMA) in the HK-2 cells were detected by RT-PCR, and the. protein expressions of TGF-beta1, COL I, and alpha-SMA were detected with Western blotting and indirect immunofluorescence. RESULTS: The urine protein from the LN patients dose-and time-dependently increased the mRNA and protein expressions of TGF-beta1, COL I, and alpha-SMA in the HK-2 cells. The TGF-beta1 mRNA level 48 h after incubation was 0.39 +/- 0.03, significantly higher than that at the beginning of incubation (0.27 +/- 0.02, P < 0.01), and the TGF-beta1 protein level 48 h after incubation was 0.37 +/- 0.03, 1.7 times that at the beginning of incubation (0.27 +/- 0.04, P < 0.01). The COL I mRNA level 48 h after incubation was 0.38 +/- 0.02, significantly higher than the baseline level (0.22 +/- 0.03, P < 0.01); and the COL I protein level 48 h after incubation was 0.44 +/- 0.03, significantly higher than the baseline level (0.19 +/- 0.02, P < 0.01). The alpha-SMA mRNA level 48 h after incubation was 0.66 +/- 0.04, significantly higher than the baseline level (0.44 +/- 0.03, P < 0.01), and the alpha-SMA protein level 48 h after incubation was 0.43 +/- 0.02, significantly higher than the baseline level (0.24 +/- 0.03, P < 0.01). CONCLUSION: Urine protein may play an important role in the renal tubular-interstitial fibrosis by inducing the production of extracellular matrix and phenotype change in HK-2 cells.