Elucidating immunological characteristics of the adenoma-carcinoma sequence in colorectal cancer patients in South Korea using a bioinformatics approach.

Colorectal cancer (CRC) is one of the top five most common and life-threatening malignancies worldwide. Most CRC develops from advanced colorectal adenoma (ACA), a precancerous stage, through the adenoma-carcinoma sequence. However, its underlying mechanisms, including how the tumor microenvironment changes, remain elusive. Therefore, we conducted an integrative analysis comparing RNA-seq data collected from 40 ACA patients who visited Dongguk University Ilsan Hospital with normal adjacent colons and tumor samples from 18 CRC patients collected from a public database. Differential expression analysis identified 21 and 79 sequentially up- or down-regulated genes across the continuum, respectively. The functional centrality of the continuum genes was assessed through network analysis, identifying 11 up- and 13 down-regulated hub-genes. Subsequently, we validated the prognostic effects of hub-genes using the Kaplan-Meier survival analysis. To estimate the immunological transition of the adenoma-carcinoma sequence, single-cell deconvolution and immune repertoire analyses were conducted. Significant composition changes for innate immunity cells and decreased plasma B-cells with immunoglobulin diversity were observed, along with distinctive immunoglobulin recombination patterns. Taken together, we believe our findings suggest underlying transcriptional and immunological changes during the adenoma-carcinoma sequence, contributing to the further development of pre-diagnostic markers for CRC.

Prediction Models for Identifying Ion Channel-Modulating Peptides via Knowledge Transfer Approaches.

Ion channels, which can be modulated by peptides, are promising drug targets for neurological, metabolic, and cardiovascular disorders. Because it is expensive and labor-intensive to experimentally screen ion channel-modulating peptides (IMPs), in-silico approaches can serve as excellent alternatives. In this study, we present PrIMP, prediction models for screening IMPs that can target sodium, potassium, and calcium ion channels, as well as nicotine acetylcholine receptors (nAChRs). To overcome the data insufficiency of the IMPs, we utilized two types of knowledge transfer approaches: multi-task learning (MTL) and transfer learning (TL). MTL enabled model training for four target tasks simultaneously with hard parameter sharing, thereby increasing model generalization. TL transferred knowledge of pre-trained model weights from antimicrobial peptide data, which was a much larger, naturally-occurring functional peptide dataset that could potentially improve the model performance. MTL and TL successfully improved the prediction performance of prediction models. In addition, a hybrid approach by implementing deep learning along with traditional machine learning was utilized, with additional performance improvements. PrIMP achieved F1 scores of 0.924 (sodium ion channel), 0.937 (potassium ion channel), 0.898 (calcium ion channel), and 0.931 (nAChRs). The pre-processed dataset and proposed model are available at https://github.com/bzlee-bio/PrIMP.

A transcriptome-wide association study of uterine fibroids to identify potential genetic markers and toxic chemicals.

Uterine fibroid is one of the most prevalent benign tumors in women, with high socioeconomic costs. Although genome-wide association studies (GWAS) have identified several loci associated with uterine fibroid risks, they could not successfully interpret the biological effects of genomic variants at the gene expression levels. To prioritize uterine fibroid susceptibility genes that are biologically interpretable, we conducted a transcriptome-wide association study (TWAS) by integrating GWAS data of uterine fibroid and expression quantitative loci data. We identified nine significant TWAS genes including two novel genes, RP11-282O18.3 and KBTBD7, which may be causal genes for uterine fibroid. We conducted functional enrichment network analyses using the TWAS results to investigate the biological pathways in which the overall TWAS genes were involved. The results demonstrated the immune system process to be a key pathway in uterine fibroid pathogenesis. Finally, we carried out chemical-gene interaction analyses using the TWAS results and the comparative toxicogenomics database to determine the potential risk chemicals for uterine fibroid. We identified five toxic chemicals that were significantly associated with uterine fibroid TWAS genes, suggesting that they may be implicated in the pathogenesis of uterine fibroid. In this study, we performed an integrative analysis covering the broad application of bioinformatics approaches. Our study may provide a deeper understanding of uterine fibroid etiologies and informative notifications about potential risk chemicals for uterine fibroid.

Nocardioides donggukensis sp. nov. and Hyunsoonleella aquatilis sp. nov., isolated from Jeongbang Waterfall on Jeju Island.

Two bacterial strains, designated MJB4T and SJ7T, were isolated from water samples collected from Jeongbang Falls on Jeju Island, Republic of Korea. Phylogenetic analysis of 16S rRNA gene sequences indicated that the two strains belonged to the genera Nocardioides and Hyunsoonleella, owing to their high similarities to Nocardioides jensenii DSM 29641T (97.5 %) and Hyunsoonleella rubra FA042 T (96.3 %), respectively. These values are much lower than the gold standard for bacterial species (98.7 %). The average nucleotide identity values between strains MJB4T, SJ7T and the reference strains, Nocardioides jensenii DSM 29641T, Nocardioides daejeonensis MJ31T and Hyunsoonleella flava T58T were 77.2, 75.9 and 75.4 %, respectively. Strains MJB4T and SJ7T and the type strains of the species involved in system incidence have average nucleotide identity and average amino acid threshold values of 60.1-82.6 % for the species boundary (95-96 %), which confirms that strains MJB4T and SJ7T represent two new species of genus Nocardioides and Hyunsoonleella, respectively. Based on phylogenetic and phenotypic data, strains MJB4T and SJ7T are considered to represent novel species of the genus Nocardioides and Hyunsoonleella, respectively, for which the names Nocardioides donggukensis sp. nov. (type strain MJB4T=KACC 21724T=NBRC 114402T) and Hyunsoonleella aquatilis sp. nov., (type strain SJ7T=KACC 21715T=NBRC 114486T) have been proposed.

A Deep Learning Approach with Data Augmentation to Predict Novel Spider Neurotoxic Peptides.

As major components of spider venoms, neurotoxic peptides exhibit structural diversity, target specificity, and have great pharmaceutical potential. Deep learning may be an alternative to the laborious and time-consuming methods for identifying these peptides. However, the major hurdle in developing a deep learning model is the limited data on neurotoxic peptides. Here, we present a peptide data augmentation method that improves the recognition of neurotoxic peptides via a convolutional neural network model. The neurotoxic peptides were augmented with the known neurotoxic peptides from UniProt database, and the models were trained using a training set with or without the generated sequences to verify the augmented data. The model trained with the augmented dataset outperformed the one with the unaugmented dataset, achieving accuracy of 0.9953, precision of 0.9922, recall of 0.9984, and F1 score of 0.9953 in simulation dataset. From the set of all RNA transcripts of Callobius koreanus spider, we discovered neurotoxic peptides via the model, resulting in 275 putative peptides of which 252 novel sequences and only 23 sequences showing homology with the known peptides by Basic Local Alignment Search Tool. Among these 275 peptides, four were selected and shown to have neuromodulatory effects on the human neuroblastoma cell line SH-SY5Y. The augmentation method presented here may be applied to the identification of other functional peptides from biological resources with insufficient data.

Methylobacterium terricola sp. nov., a gamma radiation-resistant bacterium isolated from gamma ray-irradiated soil.

A gamma radiation-resistant and pink-pigmented bacterial strain, designated as 17Sr1-39T, was isolated from a gamma ray-irradiated soil sample collected in the Republic of Korea. Cells were Gram-stain-negative, strictly aerobic, flagellated, asporogenous, rod-shaped and methylotrophic. Results of 16S rRNA gene sequence analysis showed that strain 17Sr1-39T was phylogenetically related to Methylobacterium currus PR1016AT (97.3 %), Methylobacterium aquaticum DSM 16371T (97.2 %), Methylobacterium platani PMB02T (97.0 %), Methylobacterium frigidaeris IER25-16T (96.6 %), Methylobacterium terrae 17Sr1-28T (96.6 %) and Methylobacterium organophilum JCM 2833T (93.4 %). The G+C content calculated based on the genome sequence was 70.4 mol%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain 17Sr1-39T and M. currus, M. aquaticum, M. platani, M. frigidaeris, M. terrae and M. organophilum were 77.3-89.9 and 22-38.2 %, respectively. The predominant fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The predominant quinone was ubiquinone 10 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on the data from phenotypic tests and genotypic differences between strain 17Sr1-39T and its close phylogenetic relatives, strain 17Sr1-39T represented a new species belonging to the genus Methylobacterium, for which the name Methylobacterium terricola sp. nov. (=KACC 52905T=NBRC 112874T) is proposed.

Integrative genomic and transcriptomic analysis of genetic markers in Dupuytren's disease.

BACKGROUND: Dupuytren's disease (DD) is a fibroproliferative disorder characterized by thickening and contracting palmar fascia. The exact pathogenesis of DD remains unknown. RESULTS: In this study, we identified co-expressed gene set (DD signature) consisting of 753 genes via weighted gene co-expression network analysis. To confirm the robustness of DD signature, module enrichment analysis and meta-analysis were performed. Moreover, this signature effectively classified DD disease samples. The DD signature were significantly enriched in unfolded protein response (UPR) related to endoplasmic reticulum (ER) stress. Next, we conducted multiple-phenotype regression analysis to identify trans-regulatory hotspots regulating expression levels of DD signature using Genotype-Tissue Expression data. Finally, 10 trans-regulatory hotspots and 16 eGenes genes that are significantly associated with at least one cis-eQTL were identified. CONCLUSIONS: Among these eGenes, major histocompatibility complex class II genes and ZFP57 zinc finger protein were closely related to ER stress and UPR, suggesting that these genetic markers might be potential therapeutic targets for DD.

Neuronal differentiation of human mesenchymal stem cells in response to the domain size of graphene substrates.

Graphene is a noncytotoxic monolayer platform with unique physical, chemical, and biological properties. It has been demonstrated that graphene substrate may provide a promising biocompatible scaffold for stem cell therapy. Because chemical vapor deposited graphene has a two dimensional polycrystalline structure, it is important to control the individual domain size to obtain desirable properties for nano-material. However, the biological effects mediated by differences in domain size of graphene have not yet been reported. On the basis of the control of graphene domain achieved by one-step growth (1step-G, small domain) and two-step growth (2step-G, large domain) process, we found that the neuronal differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) highly depended on the graphene domain size. The defects at the domain boundaries in 1step-G graphene was higher (×8.5) and had a relatively low (13% lower) contact angle of water droplet than 2step-G graphene, leading to enhanced cell-substrate adhesion and upregulated neuronal differentiation of hMSCs. We confirmed that the strong interactions between cells and defects at the domain boundaries in 1step-G graphene can be obtained due to their relatively high surface energy, which is stronger than interactions between cells and graphene surfaces. Our results may provide valuable information on the development of graphene-based scaffold by understanding which properties of graphene domain influence cell adhesion efficacy and stem cell differentiation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 43-51, 2018.

Inhibitory Effects of Novel SphK2 Inhibitors on Migration of Cancer Cells.

BACKGROUND: Cell migration is an essential process for survival and differentiation of mammalian cells. Numerous diseases are induced or influenced by inappropriate regulation of cell migration, which plays a key role in cancer cell metastasis. In fact, very few anti-metastasis drugs are available on the market. SphKs are enzymes that convert sphingosine to sphingosine-1-phosphate (S1P) and are known to control various cellular functions, including migration of cells. In human, SphK2 is known to promote apoptosis, suppresses cell growth, and controls cell migration; in addition, the specific ablation of SphK2 activity was reported to inhibit cancer cell metastasis. OBJECTIVE: The previously identified SG12 and SG14 are synthetic analogs of sphingoid and can specifically inhibit the functions of SphK2. We investigated the effects of the SphK2 specific inhibitors on the migratory behavior of cells. METHOD: We investigated how SG12 and SG14 affect cell migration by monitoring both cumulative and individual cell migration behavior using HeLa cells. RESULTS: SG12 and SG14 mutually showed stronger inhibitory effects with less cytotoxicity compared with a general SphK inhibitor, N,N-dimethylsphingosine (DMS). The mechanistic aspects of specific SphK2 inhibition were studied by examining actin filamentation and the expression levels of motility-related genes. CONCLUSION: The data revealed that SG12 and SG14 resemble DMS in decreasing overall cell motility, but differ in that they differentially affect motility parameters and motility-related signal transduction pathways and therefore actin polymerization, which are not altered by DMS. Our findings show that SphK2 inhibitors are putative candidates for anti-metastatic drugs.

In vitro anti-obesity effects of sesamol mediated by adenosine monophosphate-activated protein kinase and mitogen-activated protein kinase signaling in 3T3-L1 cells.

Sesamol is a phenol derivative of sesame oil and a potent anti-oxidant, anti-inflammatory, anti-hepatotoxic, and anti-aging compound. We investigated the effects of sesamol on the molecular mechanisms of adipogenesis in 3T3-L1 preadipocytes. The intracellular lipid accumulation accompanied by increased extracellular release of free glycerol was decreased during differentiation on treating 3T3-L1 with sesamol. Sesamol treatment on 3T3-L1 inhibited adipogenic differentiation by down-regulating adipogenesis-related factors (C/EBPα, PPARγ, and SREBP-1). Lipid accumulation was repressed by decreasing fatty acid synthase and by up-regulating lipolysis-response genes (HSL and LPL). The molecular mechanisms of sesamol-induced inhibition in adipogenesis were mediated by increased levels of phosphorylated adenosine monophosphate-activated protein kinase and its substrate acetyl-CoA carboxylase. Sesamol treatment, in turn, modulated the different members of the mitogenactivated protein kinase family by suppressing phosphorylation of ERK 1/2 and JNK and by increasing the phosphorylation of p38. In summary, sesamol inhibits adipogenic differentiation by reducing phosphorylation levels of ERK 1/2 and JNK while inducing lipolysis by activating p38 and AMPK. Our results demonstrate that the molecular mechanisms of in vitro anti-obesity effects of sesamol are due to the combined effects of preventing both lipid accumulation and adipogenesis.

Identification of Potential Novel Biomarkers and Signaling Pathways Related to Otitis Media Induced by Diesel Exhaust Particles Using Transcriptomic Analysis in an In Vivo System.

INTRODUCTION: Air pollutants are associated with inflammatory diseases such as otitis media (OM). Significantly higher incidence rates of OM are reported in regions with air pollution. Diesel exhaust particles (DEPs) comprise a major class of contaminants among numerous air pollutants, and they are characterized by a carbonic mixture of polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, and small amounts of sulfate, nitrate, metals and other trace elements. DEP exposure is a risk factor for inflammatory diseases. Our previous study identified potential biomarkers using gene expression microarray and pathway analyses in an in vitro system. Although in vitro investigations have been conducted to elucidate plausible biomarkers and molecular mechanisms related to DEP exposure, in vivo studies are necessary to identify the exact biological relevance regarding the incidence of OM caused by DEP exposure. In this study, we identified potential molecular biomarkers and pathways triggered by DEP exposure in a rodent model. METHODS: Transcriptomic analysis was employed to identify novel potential biomarkers in the middle ear of DEP-exposed mice. RESULTS: A total of 697 genes were differentially expressed in the DEP-exposed mice; 424 genes were upregulated and 273 downregulated. In addition, signaling pathways among the differentially expressed genes mediated by DEP exposure were predicted. Several key molecular biomarkers were identified including cholinergic receptor muscarinic 1 (CHRM1), erythropoietin (EPO), son of sevenless homolog 1 (SOS1), estrogen receptor 1 (ESR1), cluster of differentiation 4 (CD4) and interferon alpha-1 (IFNA1). CONCLUSIONS: Our results shed light on the related cell processes and gene signaling pathways affected by DEP exposure. The identified biomarkers might be potential candidates for determining early diagnoses and effective treatment strategies for DEP-mediated disorders.

Galectin-3 supports stemness in ovarian cancer stem cells by activation of the Notch1 intracellular domain.

Ovarian cancer is the most lethal gynecologic disease because usually, it is lately sensed, easily acquires chemoresistance, and has a high recurrence rate. Recent studies suggest that ovarian cancer stem cells (CSCs) are involved in these malignancies. Here, we demonstrated that galectin-3 maintains ovarian CSCs by activating the Notch1 intracellular domain (NICD1). The number and size of ovarian CSCs decreased in the absence of galectin-3, and overexpression of galectin-3 increased them. Overexpression of galectin-3 increased the resistance for cisplatin and paclitaxel-induced cell death. Silencing of galectin-3 decreased the migration and invasion of ovarian cancer cells, and overexpression of galectin-3 reversed these effects. The Notch signaling pathway was strongly activated by galectin-3 overexpression in A2780 cells. Silencing of galectin-3 reduced the levels of cleaved NICD1 and expression of the Notch target genes, Hes1 and Hey1. Overexpression of galectin-3 induced NICD1 cleavage and increased expression of Hes1 and Hey1. Moreover, overexpression of galectin-3 increased the nuclear translocation of NICD1. Interestingly, the carbohydrate recognition domain of galectin-3 interacted with NICD1. Overexpression of galectin-3 increased tumor burden in A2780 ovarian cancer xenografted mice. Increased expression of galectin-3 was detected in advanced stages, compared to stage 1 or 2 in ovarian cancer patients, suggesting that galectin-3 supports stemness of these cells. Based on these results, we suggest that targeting galectin-3 may be a potent approach for improving ovarian cancer therapy.

(-)-Epicatechin derivate from Orostachys japonicus as potential inhibitor of the human butyrylcholinesterase.

Cholinesterase inhibitors block the bioconversion of neurotransmitters by cholinesterase in the nervous system. Epicatechin derivatives (1, 3 and 5), polyphenols (6 and 7) from Orostachys japonicus, and catechin derivatives (2 and 4) from our in-house library were evaluated for their inhibitory activity on cholinesterase. Compound 5 exhibited IC50 values of 58.3±2.4 and 17.8±3.8µg/mL on AChE and BuChE, respectively. Compound 5 inhibited BuChE more strongly than AChE through a competitive behavior. In silico binding positions of 5 in the active site were predicted using Autodock 4.2 and processed in a 10000-ps molecular dynamics simulation to assess the stability of compound 5 binding.

Effect of upregulated TLR2 expression from G-CSF-mobilized donor grafts on acute graft-versus-host disease.

Our previous study demonstrated that G-CSF treatment increased the expression of TLR2 in donor grafts; this contributed to rapid engraftment after allogeneic hematopoietic stem cell transplantation (HSCT) in mice. In the current study, we investigated the effects of upregulated TLR2 expression in G-CSF-mobilized donor grafts on acute graft-versus-host disease (GVHD). We found that TLR2 was highly expressed on myeloid cell populations but not T and B cells from the spleens of G-CSF-treated donor mice. After transplantation, the mortality and disease severity in recipients were not significantly different between G-CSF-treated TLR2-/- and wt donor grafts. Although endogenous TLR2 ligand was detected in the serum of both recipients, T cells from TLR2-/- and wt donors have the same ability regarding alloreactivity. Moreover, the blockade of TLR2 signaling in recipients by administering anti-TLR2 blocking antibody after BMT did not lead to a significant difference in acute GVHD compared with control IgG treatment. However, the hematopoietic ability of G-CSF-mobilized lin−c-kit+ HSCs from TLR2-/- donor grafts was lower than that from wt donor grafts. Our results demonstrate that upregulated TLR2 expression in G-CSF-mobilized donor grafts has no effect on acute GVHD, suggesting that TLR2 is a valuable target for increasing HSCT efficiency in order to enhance engraftment without exacerbating acute GVHD.

A novel adamantyl benzylbenzamide derivative, AP736, suppresses melanogenesis through the inhibition of cAMP-PKA-CREB-activated microphthalmia-associated transcription factor and tyrosinase expression.

Melanogenesis is essential for the protection of skin against UV, but excessive production of melanin causes unaesthetic hyperpigmentation. Much effort is being made to develop effective depigmenting agents. Here, we found that a tyrosinase inhibitor, AP736 (5-adamantan-1-yl-N-(2,4-dihydroxy-benzyl)-2,4-dimethoxy-benzamide) potently suppresses tyrosinase expression, and the mechanism underlying was elucidated. AP736 attenuated the melanin production induced by diverse melanogenic stimuli in murine and human melanocytes. It suppressed the expression of key melanogenic enzymes; tyrosinase, tyrosinase-related protein-1 and tyrosinase-related protein-2. The expression of microphthalmia-associated transcription factor (MiTF), a major promoter of melanogenesis was also decreased. AP736 inhibited the activation of cAMP response element-binding protein (CREB) and phosphokinase A (PKA), and cAMP elevation, reflecting that cAMP-PKA-CREB signalling axis was suppressed, resulting in the downregulation of MiTF and tyrosinase. Along with the previously reported tyrosinase inhibitory activity, the suppression of cAMP-PKA-CREB-mediated MiTF and tyrosinase expression by AP736 may be efficient for the treatment for hyperpigmentation.

Endogenous gastric-resident mesenchymal stem cells contribute to formation of cancer stroma and progression of gastric cancer.

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) contribute to carcinogenesis and cancer progression, although their origin and role remain unclear. We recently identified and investigated the in situ identity and implications of gastric submucosa-resident mesenchymal stem cells (GS-MSCs) in the progression of gastric carcinogenesis. METHODS: We isolated GS-MSCs from gastric submucosa using hydrogel-supported organ culture and defined their identity. Isolated cells were assessed in vitro by immunophenotype and mesengenic multipotency. Reciprocal interactions between GS-MSCs and gastric cancer cells were evaluated. To determine the role of GS-MSCs, xenografts were constructed of gastric cancer cells admixed with or without GS-MSCs. RESULTS: Isolated cells fulfilled MSCs requirements in regard to plastic adherence, stromal cell immunophenotype, and multipotency. We demonstrated a paracrine loop that gastric cancer cells enhanced the migration, proliferation, and differentiation of GS-MSCs; additionally, GS-MSCs promoted the proliferation of gastric cancer cell in vitro. Xenograft experiments showed that GS-MSCs significantly promoted cancer growth and angiogenesis. GS-MSCs that integrated into gastric cancer became not only CAFs but also rarely endothelial cells which contributed to the formation of cellular and vascular cancer stroma. CONCLUSIONS: Endogenous GS-MSCs play an important role in gastric cancer progression.

Mussel-inspired immobilization of vascular endothelial growth factor (VEGF) for enhanced endothelialization of vascular grafts.

Most polymeric vascular prosthetic materials have low patency rate for replacement of small diameter vessels (<5 mm), mainly due to failure to generate healthy endothelium. In this study, we present polydopamine-mediated immobilization of growth factors on the surface of polymeric materials as a versatile tool to modify surface characteristics of vascular grafts potentially for accelerated endothelialization. Polydopamine was deposited on the surface of biocompatible poly(L-lactide-co-ε-caprolactone) (PLCL) elastomer, on which vascular endothelial growth factor (VEGF) was subsequently immobilized by simple dipping. Surface characteristics and composition were investigated by using scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Immobilization of VEGF on the polydopamine-deposited PLCL films was effective (19.8 ± 0.4 and 197.4 ± 19.7 ng/cm(2) for DPv20 and DPv200 films, respectively), and biotin-mediated labeling of immobilized VEGF revealed that the fluorescence intensity increased as a function of the concentration of VEGF solution. The effect of VEGF on adhesion of HUVECs was marginal, which may have been masked by polydopamine layer that also enhanced cell adhesion. However, VEGF-immobilized substrate significantly enhanced proliferation of HUVECs for over 7 days of in vitro culture and also improved their migration. In addition, immobilized VEGF supported robust cell to cell interactions with strong expression of CD 31 marker. The same process was effective for immobilization of basic fibroblast growth factor, demonstrating the robustness of polydopamine layer for secondary ligation of growth factors as a simple and novel surface modification strategy for vascular graft materials.

Low level of lead can induce phosphatidylserine exposure and erythrophagocytosis: a new mechanism underlying lead-associated anemia.

Anemia is probably one of the most well-known toxic effects of lead. Previously, lead-induced anemia was considered to be from the inhibition of δ-aminolevulinic acid dehydratase participating in the heme biosynthesis. However, little is known whether lead could affect the destruction of erythrocyte, another important factor for anemia. In the present study, we demonstrated that lead could accelerate the splenic sequestration of erythrocytes through phosphatidylserine (PS) exposure and subsequently increased erythrophagocytosis. In freshly isolated human erythrocytes, Pb(2+)- induced PS exposure at relatively low concentrations (∼0.1 µM) by inhibiting flippase, a key aminophospholipid translocase for the maintenance of PS asymmetry and adenosine triphosphate depletion appeared to underlie this phenomenon. Abnormal shape changes of erythrocytes and microvesicle generation and other triggers for the erythrophagocytosis were also observed in the Pb(2+)-exposed erythrocytes. In vitro data showed that human macrophage indeed recognized and phagocytosis PS-exposed erythrocytes. In good accordance with these in vitro results, the oral administration of Pb(2+) increased PS exposure on erythrocytes in rat in vivo. In addition, reduction of hematocrit and hemoglobin and increased spleen weight were observed along with enhanced splenic sequestration of erythrocytes in the rats exposed to Pb(2+) subchronically for 4 weeks through drinking water. In conclusion, these results suggest that Pb(2+)-induced anemia may be explained at least in part by increased PS exposure on erythrocytes, erythrophagocytosis, and splenic sequestration.

Cell density sensing and size determination.

The social amoeba Dictyostelium discoideum is one of the leading model systems used to study how cells count themselves to determine the number and/or density of cells. In this review, we describe work on three different cell-density sensing systems used by Dictyostelium. The first involves a negative feedback loop in which two secreted signals inhibit cell proliferation during the growth phase. As the cell density increases, the concentrations of the secreted factors concomitantly increase, allowing the cells to sense their density. The two signals act as message authenticators for each other, and the existence of two different signals that require each other for activity may explain why previous efforts to identify autocrine proliferation-inhibiting signals in higher eukaryotes have generally failed. The second system involves a signal made by growing cells that is secreted only when they starve. This then allows cells to sense the density of just the starving cells, and is an example of a mechanism that allows cells in a tissue to sense the density of one specific cell type. The third cell density counting system involves cells in aggregation streams secreting a signal that limits the size of fruiting bodies. Computer simulations predicted, and experiments then showed, that the factor increases random cell motility and decreases cell-cell adhesion to cause streams to break up if there are too many cells in the stream. Together, studies on Dictyostelium cell density counting systems will help elucidate how higher eukaryotes regulate the size and composition of tissues.

Low-level mercury can enhance procoagulant activity of erythrocytes: a new contributing factor for mercury-related thrombotic disease.

BACKGROUND: Associations between cardiovascular diseases and mercury have been frequently described, but underlying mechanisms are poorly understood. OBJECTIVES: We investigate the procoagulant activation of erythrocytes, an important contributor to thrombosis, by low-level mercury to explore the roles of erythrocytes in mercury-related cardiovascular diseases. METHODS: We used freshly isolated human erythrocytes and ex vivo and in vivo thrombosis models in rats to investigate mercury-induced procoagulant activity. RESULTS: Prolonged exposure to low-dose mercuric ion (Hg(2+); 0.25-5 microM for 1-48 hr) induced erythrocyte shape changes from discocytes to echinocytes to spherocytes, accompanied by microvesicle (MV) generation. These MVs and remnant erythrocytes expressed phosphatidylserine (PS), an important mediator of procoagulant activation. Hg(2+) inhibited flippase, an enzyme that recovers PS into the inner leaflet of the cell membrane, and activated scramblase, an enzyme that alters lipid asymmetry in the cell membrane. Consistent with these activity changes, Hg(2+) increased intracellular calcium and depleted ATP and protein thiol. A thiol supplement reversed Hg(2+)-induced MV generation and PS exposure and inhibited the increase in calcium ion (Ca(2+)) and depletion of ATP, indicating that free-thiol depletion was critical to Hg(2+)-mediated procoagulant activity. The procoagulant activity of Hg(2+)-treated erythrocytes was demonstrated by increased thrombin generation and endothelial cell adhesion. We further confirmed Hg(2+)-mediated procoagulant activation of erythrocytes in ex vivo and in vivo rat thrombosis models, where Hg(2+) treatment (0.5-2.5 mg/kg) increased PS exposure and thrombus formation significantly. CONCLUSION: This study demonstrated that mercury could provoke procoagulant activity in erythrocytes through protein-thiol depletion-mediated PS exposure and MV generation, ultimately leading to enhanced thrombosis.