Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.     

Renaturation of formyltetrahydrofolate synthetase from urea and guanidinium chloride solutions

Formyltetrahydrofolate synthetase from Clostridium cylindrosporum and Clostridium acidi-urici was denatured in 6 M urea and 4 M guanidinium chloride. Viscometric, fluorimetric and ultracentrifugal measurements were used to determine that the protein is completely unfolded under these conditions. The polypeptide chains refold upon dilution of the denaturant-protein solutions to give final concentrations of 0.5 M urea or 0.1 M guanidinium chloride. In the presence of NH4+, but not in its absence, the refolded proteins associate to produce the catalytically active tetramer. Refolding and reassociation were followed by measuring changes in protein fluorescence and by determination of sedimentation constants. Under most conditions 80% of the enzymic activity is recovered.     

Cigarette Smoke Stimulates SARS-CoV-2 Internalization by Activating AhR and Increasing ACE2 Expression in Human Gingival Epithelial Cells

A large body of evidence shows the harmful effects of cigarette smoke to oral and systemic health. More recently, a link between smoking and susceptibility to coronavirus disease 2019 (COVID-19) was proposed. COVID-19 is due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which uses the receptor ACE2 and the protease TMPRSS2 for entry into host cells, thereby infecting cells of the respiratory tract and the oral cavity. Here, we examined the effects of cigarette smoke on the expression of SARS-CoV-2 receptors and infection in human gingival epithelial cells (GECs). We found that cigarette smoke condensates (CSC) upregulated ACE2 and TMPRSS2 expression in GECs, and that CSC activated aryl hydrocarbon receptor (AhR) signaling in the oral cells. ACE2 was known to mediate SARS-CoV-2 internalization, and we demonstrate that CSC treatment potentiated the internalization of SARS-CoV-2 pseudovirus in GECs in an AhR-dependent manner. AhR depletion using small interference RNA decreased SARS-CoV-2 pseudovirus internalization in CSC-treated GECs compared with control GECs. Our study reveals that cigarette smoke upregulates SARS-CoV-2 receptor expression and infection in oral cells. Understanding the mechanisms involved in SARS-CoV-2 infection in cells of the oral cavity may suggest therapeutic interventions for preventing viral infection and transmission.     

Classification and interactive segmentation of EEG synchrony patterns

This paper presents a novel methodology for the exploratory analysis of power and synchronization patterns in EEG data from psychophysiological experiments. The methodology is based on the segmentation of the time-frequency plane in regions with relatively homogeneous synchronization patterns, which is performed by means of a seeded region-growing algorithm, and a Bayesian regularization procedure. We have implemented these methods in an interactive application for the study of cognitive experiments, although some of the techniques discussed in this work can also be applied to other multidimensional data sets. To demonstrate our methodology, results corresponding to a figure and word categorization EEG experiment are presented.     

Evolution of the properties of a poly(l‐lactic acid) scaffold with double porosity during in vitro degradation in a phosphate‐buffered saline solution

A poly(l ‐lactic acid) scaffold prepared by a combination of freeze‐extraction and porogen‐leaching methods was submitted to static degradation in a phosphate‐buffered saline solution at pH 7.4 and 37°C for up to 12 months. After 6 months of degradation, the scaffold maintained its integrity, although noticeable changes in its permeability and pore size were recorded. After 12 months, scanning electron microscopy pictures showed that most of the trabeculae were broken, and the sample disaggregated under minimum loading. Neither weight loss nor crystallinity changes in the first heating calorimetric scan were observed during the degradation experiment. However, after 12 months, a rise in the crystallinity from 13 to 38% and a drop in the glass‐transition temperature from 58 to 54°C were measured in the second heating scan. The onset of thermal degradation moved from 300 to 210°C after 12 months. Although the elastic modulus suffered only a very slight reduction with degradation time, the aggregate modulus decreased 44% after 6 months. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40956.     

Heparin releases newly synthesized cell surface-associated apolipoprotein E from HepG2 cells

Heparin significantly increased the amount of newly synthesized apolipoprotein E (apoE) released by HepG2 cells. Culturing cells in the presence of 10 micrograms/ml of heparin for 2-6 days caused a 1.3-fold (day 2) to 3-fold (day 6) increase of extracellular apoE without affecting the total amount of apoE synthesized by the cells. The amounts of apoA-I and apoB produced by HepG2 cells were unaffected by heparin. Surprisingly, short-term treatment with heparin (15-30 min) also increased extracellular apoE by 2- to 3-fold. In this situation, heparin exerted its effect on apoE post-translationally. Among glycosaminoglycans (GAGs), only heparan sulfate mimicked heparin at a concentration of 10 micrograms/ml; hyaluronic acid and the chondroitin sulfates were effective only at a higher concentration (100 micrograms/ml). Extracellular apoE was not increased by treating cells with anti-apoE antiserum or a heparin-binding peptide of apoE (amino acids 130-169). Removal of cell surface-associated GAGs by culturing cells in 4-methylumbelliferyl-beta-D-xyloside ablated the effect of heparin on apoE. ApoE was released from cells by treatment with heparinases I and III, but not by chondroitinase ABC. The results provide evidence that a heparin-releasable pool of newly synthesized apoE is associated with cell surface GAGs that resemble heparin and/or heparan sulfate.     

Cholesteryl ester analogs inhibit cholesteryl ester but not triglyceride transfer catalyzed by the plasma cholesteryl ester-triglyceride transfer protein

A lipid transfer protein complex (LTC), purified from human plasma by immunoaffinity chromatography, catalyzed the interlipoprotein transfer of cholesteryl esters (CE) and triglycerides (TG). The CE transfer activity of LTC was governed by the strucutre of the CE. Incubation of LTC with long chain CE both activated and stabilized LTC. Short chain CE also enhanced the CE and TG transfer activity of LTC during the initial time of incubation. However, LTC's incubation with short chain CE induced a subsequent and time-dependent loss of CE transfer activity without concomitant loss of TG transfer activity. The data indicate that the CE and TG transfer activity of LTC can be reglated independently.     

Los Angeles housing models and neighbourhoods’ role in supportive housing residents’ social integration

Social integration is an indicator of programmatic success in supportive housing, yet is an ongoing challenge for residents. This study examines varying supportive housing models’ (i.e. congregate, single-site, scatter-site) and neighborhoods’ (i.e. Skid Row, Downtown Los Angeles [DTLA], Other) differential impact on social integration outcomes- measured by residents’ social networks (i.e. size, diversity, social support). Participants were formerly homeless English or Spanish speaking unaccompanied adults (N=405), aged 39 years or older, living in supportive housing for 3 months. Housing model and neighborhood were examined separately with social network measures in controlled multivariable linear regression models. Compared to Skid Row residents, DTLA residents reported less emotional support and less tangible support, while residents in Other neighborhoods reported less emotional support and less instrumental support. Findings suggest overall differing housing models may be less influential in social integration, while neighborhoods may facilitate social support.     

Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.