Quantitative proteomics reveals TMOD1-related proteins associated with water balance regulation.

The distal tubule and collecting duct in kidney regulate water homeostasis. TMOD1 is an actin capping protein that plays an important role in controlling the organization of actin filaments. In this study, we found TMOD1 was specifically expressed in distal tubules and collecting ducts. To investigate the role of TMOD1, we created Tmod1flox/flox mice and bred them with Ksp-Cre mice to generate tubule-specific Tmod1 knockout mice, Tmod1flox/flox/Ksp-Cre+ (designated as TFK). As compared with control mice, TFK mice showed oliguria, hyperosmolality urine, and high blood pressure. To determine the mechanisms underlying this phenotype, we performed label-free quantitative proteomics on kidneys of TFK and control mice. Total of 83 proteins were found differentially expressed. Bioinformatic analysis indicated that biological processes, including protein phosphorylation and metabolic process, were involved in TMOD1 regulatory network. Gene set enrichment analysis showed that multiple pathways, such as phosphatidylinositol signaling system and GnRH signaling pathway, were strongly associated with Tmod1 knockout. Western blot validated the down-regulation of three proteins, TGFBR2, SLC25A11, and MTFP1, in kidneys of TFK mice. Our study provides valuable information on the molecular functions and the regulatory network of Tmod1 gene in kidney, as well as the new mechanisms for the regulation of water balance.

Fluid Shear Stress Upregulates E-Tmod41 via miR-23b-3p and Contributes to F-Actin Cytoskeleton Remodeling during Erythropoiesis.

The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm2) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3'UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, PE0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and PE0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling.

The effects of non-ionic contrast medium on the hemorheology in vitro and in vivo.

Contrast media are the commonly used agents in radiology. However, because of their characteristics of high osmolality, high viscosity, and chemical toxicity, the administrations of contrast media have been shown to cause adverse effects especially on hemorheology in short time course. The present study is to find the effects of a non-ionic contrast medium, iopromide, on the hemorheology in long time course both in vitro and in vivo. For in vitro treatment, human peripheral blood samples were incubated with contrast medium at 37°C for 0.5, 1 and 2 h. For in vivo study, about 15 ml of contrast medium was injected into rabbits and blood samples were collected at 0.5, 2, 6, and 24 h after the bolus injection. Hemorheological parameters were examined. Results showed that hematocrit adjusted whole blood viscosity increased significantly at 1 h after in vitro treatment of contrast medium, while it decreased at 0.5 h and remained low till 6 h after bolus injection. Ektacytometer showed that erythrocyte deformability decreased to the lowest level at 2 h in vitro and it dropped at 0.5 h and resumed to normal after 2 h in vivo. Erythrocyte small deformation indices were reduced by contrast medium in both in vitro and in vivo studies. Erythrocyte orientation index was also reduced in in vivo study. Erythrocyte electrophoresis rates at all time points decreased but osmotic fragility did not change in both studies. These impaired hemorheological parameters may disturb the microcirculation and cause adverse effects in patients with kidney diseases.

[Quantitative calculation of drugs distribution parameter in the brain extracellular space by using MRI tracer].

OBJECTIVE: To build a mathematical model to simulate the drug distribution accompanying with diffusion, distribution and clearance in the brain extracellular space (ECS). METHODS: Magnetic resonance imaging (MRI) technology was used to monitor changes in the signal-intensity-related tracer gadolinium-diethylene triamine pentaacetic acidm(Gd-DTPA), as an external drug which was injected into the rat brain, and then the mathematical model was built by using the data to establish the diffusion, distribution and clearance process of Gd-DTPA in the brain ECS. The model equation was resolved by Laplace transform. In the sphere coordinates, the linear regressive model was adopted to obtain the estimation method of diffusion coefficient, clearance rate of drugs distribution in the brain ECS. RESULTS: The diffusion coefficient D and the clearance rate k were obtained as (2.73±0.364)×10(-4) mm(2)/s and (1.40±0.206)×10(-5) /s, respectively. CONCLUSION: The proposed method can accurately reflect the isotropic drug distribution in the brain ECS, and can serve as the foundation to further solve problems about the orthotropic distribution in the brain ECS.

Effects of Gekko sulfated polysaccharide-protein complex on the defective biorheological characters of dendritic cells under tumor microenvironment.

We previously isolated a sulfated polysaccharide-protein complex from Gekko swinhonis Guenther, a traditional Chinese medicine, and have demonstrated its direct anti-cancer effect on human hepatocellular carcinoma cell line SMMC-7721. Here we investigated the effects of Gekko sulfated polysaccharide-protein complex (GSPP) on the defective biorheological characters of dendritic cells (DCs) under SMMC-7721 microenvironment. Our findings have shown that the biorheological properties of DCs were severely impaired by SMMC-7721 microenvironment, including decreased cell deformability, migration, and electrophoresis mobility, increased osmotic fragilities, and changed organizations of cytoskeletal proteins. We also found decreased secretion of interleukin (IL)-12 and increased secretion of IL-10 in DCs. However, supernatant collected from nonmalignant liver cells had no effect on these parameters. SMMC-7721 cells were treated with GSPP and the supernatant was used to culture DCs. We found that the defective biorheological parameters of DCs, except for osmotic fragility, were partially or completely improved. The secretion of IL-12 did not change as compared with that of DCs in SMMC-7721 microenvironment, but the secretion of IL-10 was resumed to the control level. Our results indicate that GSPP could partially restore the defective biorheological characteristics of DCs via modifying the tumor microenvironment and decreasing the secretion of IL-10 of DCs.

Knockdown of hTERT alters biophysical properties of K562 cells resulting in decreased migration rate in vitro.

It has been shown that 90% of tumors, including hematological malignant tumors and leukemia, have much higher levels of telomerase expression than normal cells. To investigate the effect of telomerase on leukemia cells, we transfected K562, a human erythroleukemia cell line with an antisense-hTERT (human telomerase reverse transcriptase) cDNA vector, and examined the biological and biophysical properties of the stably transfected cells (referred to as KAT). Un-transfected cells (K562) and cells transfected with the empty vector (referred to as KC) were used as controls. Cell growth curve and (3)H-TdR test showed that the growth rate and DNA synthesis of KAT decreased compared with those of K562 and KC cells. Apoptosis and cell cycle distribution in KAT cells under normal culture condition were similar to those of K562 and KC cells, but changed after serum deprivation. KAT cells had significantly different biophysical characteristics from K562 and KC in terms of cell electrophoresis, membrane fluidity, membrane fluidity, and viscoelasticity. Furthermore, the transendothelial migration rate of KAT was much lower than those of K562 and KC cells. Confocal microscopy showed that KAT cells had higher F-actin content, suggesting the reorganization of cytoskeleton. Flow cytometry analysis revealed a lowered intracellular calcium concentration and CD71 expression, explaining the high F-actin content in KAT cells. In conclusion, we found that the knockdown of hTERT in K562 cells changed their cytoskeleton and biophysical features, and reduced the cell migration.

Biorheological changes of dendritic cells at the different differentiation stages.

The differentiation, maturation and functioning of Dendritic cells (DCs) are dynamic processes. This study investigated the changes of DCs' migration ability and biorheological properties during their differentiation. Transmigration assay showed that, DCs' migration rate was improved significantly as they differentiate (p < 0.05); NSC (Rac1 blocker) treatment could significantly decrease their migration rates (p < 0.05). Confocal images showed that, F-actin uniformly distributed in monocytes; with DC's differentiation, F-actin began to remodel and gather at the site of dendrites; the images presented surface ruffles and uneven sawtooth-like cytoskeletal structures. Fluorescence polarization analysis showed that, membrane fluidity was increased significantly with DC's differentiation (p < 0.05). CD62L was upregulated significantly (p < 0.05) on the third and ninth days. CD2 was upregulated significantly (p < 0.05) until the seventh day. DC's electrophoretic mobility was increased continuously, especially increased significantly from the third day to the fifth day and the final stage (p < 0.05). These results indicate that there are significant changes in the biorheological properties of DCs during their differentiation.

Biomechanical alterations of dendritic cells by co-culturing with K562 CML cells and their potential role in immune escape.

Dendritic cells (DCs), which are potent antigen presenting cells (APCs), are utilized to deliver the signals essential for the initiation of immune responses. In this study, we used an interdisciplinary approach to characterize the effect of K562 cells, a human chronic myeloid leukemia (CML) cell line, on the biomechanical characteristics and immune functions of DCs. When co-cultured with K562 cells, the biomechanical and immunological characteristics of immature DCs (imDCs) and mature DCs (mDCs) were severely impaired compared with controls. The changes include increased membrane viscoelasticity, reorganized cytoskeleton (F-actin), suppressed capability of antigen uptake, transendothelium migration, and activation of naïve T cells. In exploring the mechanisms of these changes, we identified several genes and proteins by microarray analysis and 2D gel electrophoresis. Changes were found in the cytoskeleton-related genes and proteins (such as cofilin1 and profilin1) and matrix-related genes and proteins (such as TIMP1 and MMP9). These findings provide a molecular basis for the biomechanical and immunological changes of DCs in response to K562 and may help to elucidate the mechanism for tumor immune escape.

Acute dichlorvos poisoning induces hemorheological abnormalities in rabbits via oxidative stress.

Dichlorvos is an important insecticide used largely. Some studies have demonstrated that organophosphate pesticide has effects on erythrocyte membrane structures, which is critical to erythrocyte function and hemorheology. The aim of the present study was to explore the effect of oxidative stress on hemorheological changes during dichlorvos poisoning in rabbits. Data indicated that after dichlorvos exposure the hematocrit adjusted viscosity at high shear rate increased and erythrocyte membrane fluidity decreased. Data obtained from plasma showed that lipid peroxidative substance-malonaldehyde was elevated and superoxide dismutase was reduced. In summary, oxidative stress does occur in dichlorvos poisoning and may lead to hemorheological alterations. The changes of hemorheology may be responsible for the pathophysiology of the dichlorvos poisoning.

Effects of Gekko sulfated polysaccharide-protein complex on human hepatoma SMMC-7721 cells: inhibition of proliferation and migration.

AIM OF THE STUDY: Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Here we investigated the structural characterization and anti-cancer effects of sulfated polysaccharide-protein complex (GSPP) isolated from Gekko swinhonis Guenther. MATERIALS AND METHODS: The structure of GSPP was characterized by high performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, beta-elimination reaction, and NMR spectroscopy. SMMC-7721 cells were used to assess the influence of GSPP on hepatocellular carcinoma. Cell proliferation and survival was determined by trypan blue exclusion assay. Cell migration was performed by wound-healing and transwell assay. The secretion of IL-8 was detected by an enzyme-linked immunosorbent assay kit. Flow cytometry was used to analyze intracellular calcium concentration, as well as cell cycle distribution and apoptosis. Confocal microscopy was used to assess the localization and configuration of actin filaments. RESULTS: GSPP was chemically characterized as a sulfated polysaccharide-protein complex with O-glycopeptide linkages. Our results showed that GSPP inhibited the proliferation of SMMC-7721 cells and blocked cells in the S phase. No direct toxicity against cells was observed. Furthermore, GSPP inhibited the migration of SMMC-7721 cells with the reduction of intracellular calcium. Actin filaments were polymerized and accumulated in the cytoplasm of the treated cells, whereas the secretion of IL-8 was not significantly changed after GSPP exposure. CONCLUSION: We describe an identified sulfated polysaccharide-protein complex, and demonstrate its direct effect on hepatocellular carcinoma cell migration via calcium-mediated regulation of the actin cytoskeleton reorganization.

Ryanodine receptor 1 mediates Ca2+ transport and influences the biomechanical properties in RBCs.

Ryanodine receptors (RyRs) are a family of Ca2+ channel proteins that mediate the massive release of Ca2+ from the endoplasmic reticulum into the cytoplasma. In the present study, we manipulated the incorporation of RyR1 into RBC membrane and investigated its influences on the intracellular Ca2+ ([Ca2+](in)) level and the biomechanical properties in RBCs. The incorporation of RyR1 into RBC membranes was demonstrated by both immunofluorescent staining and the change of [Ca2+](in) of RBCs. In the presence of RyR1, [Ca2+](in) showed biphasic changes, i.e., it increased with the extracellular Ca2+ ([Ca2+](ex)) up to 5muM and then decreased with the further increase of [Ca2+](ex). However, [Ca2+](in) remained constant in the absence of the RyR1. The results of biomechanical measurements on RBCs, including deformability, osmotic fragility, and membrane microviscosity, reflected similar biphasic changes of [Ca2+](in) mediated by RyR1 with the increases of [Ca2+](ex). Therefore, it is believed that RyR1 can incorporate into RBC membrane in vitro, and mediate Ca2+ influx, and then regulate RBC biomechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca2+ release channel.

Effects of myakuryu on hemorheological characteristics and mesenteric microcirculation of rats fed with a high-fat diet.

There is evidence that hyperlipidemia can induce hemorheological and microcirculatory disturbances. Myakuryu, a Chinese traditional medicine is efficacious in promoting lipid metabolism and protecting oxidative stress, but whether this drug can ameliorate rheologic disturbances caused by hyperlipidemia is still unknown. The present study was conducted to investigate the effects of myakuryu on hemorheological and microcirculatory disturbances induced by hyperlipidemia. Wistar rats were divided into a group on control diet (n=8) and a group on high-fat diet (HFD, n=44). Eight weeks later, plasma triglyceride (TG) and total cholesterol (TC) were determined. Sixteen animals with the highest levels of hyperlipidemia from the HFD group were randomly divided into two sub-groups: the untreated hyperlipidemia group (n=8) and the group treated with myakuryu (n=8). At the end of the sixteenth week, rheological and microcirculatory parameters were measured. Chemical analysis showed that myakuryu treatment caused significant reductions of plasma TG and TC levels (P<0.01), and the cholesterol/phospholipid ratio in the erythrocyte membrane (P<0.05). Rheological and microcirculatory measurements showed that myakuryu treatment led to a significant decrease in the erythrocyte aggregation index, plasma viscosity and blood viscosity at shear rates of 50, 100 and 150 s(-1) and in adherent leukocytes in mesenteric venules. There was a significant increase in erythrocyte deformation, electrophoretic mobility, membrane fluidity and F-actin content in the erythrocyte membrane as well as in red cell velocity in mesenteric venules. Our findings suggest that myakuryu treatment can improve blood flow and reduce adherent leukocytes in the venules of rats fed with HFD by ameliorating blood viscosity, erythrocyte deformability and aggregation, and other hemorheological characteristics.

Synergistic effect of cytokines EPO, IL-3 and SCF on the proliferation, differentiation and apoptosis of erythroid progenitor cells.

Erythroblasts were obtained from murine spleen. After cultured for 12 hr, the cells were divided into four groups with the use of the following cytokines in culture: EPO, EPO+SCF, EPO+IL-3, and EPO+IL-3+SCF. Cell proliferation assay was done. Apoptosis rates were obtained by using a flow cytometer. Mitochondrial membrane potential (MMP) was assessed in flow cytometry (FCM) by labeling with rhodamine 123. Mitochondrial enzyme activity (MEA) was evaluated with MTT colorimetric assay. The cells were labeled with Fluo-3/Am Ester and Ca(2+) concentration was measured. The expression of Bax mRNA and Bcl-2 mRNA was analyzed by RT-PCR. At same time, the expression of Bax and Bcl-2 was analyzed by western blotting. Our results showed that IL-3 and SCF have synergistic effects with EPO on the proliferation, differentiation and apoptosis of erythroid progenitors.

Exogenous wild-type p53 gene improved survival of nude mice injected with murine erythroleukemia cell line through amelioration of hemorheological changes.

OBJECTIVES: Previous investigations have shown that human wild-type p53 gene (WTp53) inhibits the growth of leukemia and tumor cells in vitro. In the present study, the authors used nude mice and examined the therapeutic role of p53 gene for erythroleukemia in vivo in the absence of MHC effects. METHODS: The nude mice were injected with murine erythroleukemia cells (MEL), MEL cells transfected with wild-type p53 gene (MEL-W), and MEL cells transfected with mutated p53 gene (MEL-M). Abnormalities were found in the hemorheological and biophysical properties of red blood cells in all 3 groups of animals, but the abnormalities were lesser in degree and later in appearance in MEL-W group than in MEL and MEL-M groups. Furthermore, the nude mice in MEL-W group lived longer than those in MEL and MEL-M groups. RESULTS: The results showed that WTp53 restrained the growth of erythroleukemia cells in vivo and reduced the erythroleukemia tumorigenesis in the microcirculation by improving the hemorheological and biophysical properties of MEL cells, which helped to prolong the life span of nude mice suffering from erythroleukemia. CONCLUSION: These results contribute to our knowledge on the use of wild-type p53 gene for the treatment of erythroleukemia disease.

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL.

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorheological indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.

Tumor-derived factors impaired motility and immune functions of dendritic cells through derangement of biophysical characteristics and reorganization of cytoskeleton.

The generation and progress of tumors are accompanied with a marked suppression of human immune system. To explore the mechanisms by which tumors escape from immune recognition, we studied the influences of tumor microenvironment on differentiation of dendritic cells (DCs), which play an important role in tumor immunology, by biophysical and immunological methods. It was found that the cytokines derived from tumors caused an increase in osmotic fragility and a decrease in membrane fluidity of DCs, disordering and elevated expression levels of cytoskeleton, and changes of the gene transcriptional levels and energy status of the cells. Moreover, IL-12 production and the expression levels of some surface-marker molecules were also suppressed. These changes led to impaired capabilities of antigen uptake, cell motility and naïve T cell activation; the abnormal biophysical characteristics of DCs may be one aspect of the immune escape mechanism of tumor. These results provide insights into the importance of the reconstruction of tumor microenvironment for immunotherapy based on the anti-cancer activities of DCs.

Biochemical and biophysical studies on the precursor cells of mouse erythrocytes at different stages.

The aim of this research is to study the biochemical and biophysical properties of the precursor cells of mouse erythrocytes at different stages and the molecular mechanisms of their regulation. We investigated the degree of terminal differentiation of splenic erythroblasts obtained from mice during the acute phase of disease caused by the anemia-inducing FVAstrain of Friend virus. We analyzed the transcription and protein levels of alpha-globin, beta-globin (erythroid special protein) and GATA-1 (a special erythroid transcription factor). We also have examined the Ca2+ concentration, the distribution and amount of F-actin, important cellular components such as nucleic acids, lipids, and proteins, and the adhesion of precursor cells of RBC at different stages to vascular endothelium. Our results indicated that Ca2+ concentration and the distribution and structure of F-actin changed with the development of proerythroblasts, and that the adhesion rate between the precursor cells and endothelial cells can be correlated with the expression levels of ICAM-1 and P-selectin. These alternations caused changes in biophysical properties of the cell, such as membrane fluidity and deformability.

TRAIL gene reorganizes the cytoskeleton and decreases the motility of human leukemic Jurkat cells.

TRAIL can selectively induce rapid apoptosis of various types of tumor cells. We induced the expression of TRAIL in Jurkat cells, and measured the adhesion of cells to human umbilical vein endothelial cells (HUVECs) and laminin (LN) in a parallel plate flow chamber system and by using a colorimetric method. The apoptosis percentage, cycle distribution, intracellular Ca(2+) concentration, and adhesion molecule expression of the cells were detected by flow cytometry. Cytoskeleton was observed with a laser confocal microscopy. The roles of adhesion molecules in the cell interaction was defined by their function blocking. The results showed that TRAIL attenuated the adhesion of Jurkat cells to HUVECs and LN, as well as their transendothelial migration. The increased apoptosis and G1-phase cell percentages, decreased intracellular Ca(2+) concentration, depolymerized actin and impaired cell deformability could contribute to the decreased adhesion of Jurkat cells caused by TRAIL. Furthermore, CD11a was found to play a more important role than CD62L in the adhesion of Jurkat cells to HUVECs. These findings contribute to the knowledge on the role of TRAIL in tumor metastasis and provide mechanistic basis for the clinical application of TRAIL and tumor therapy.

Biophysical studies on the differentiation of human CD14+ monocytes into dendritic cells.

Dendritic cells (DCs), which are the most efficient antigen-presenting cells (APCs) currently known, can be derived from CD14+ monocytes (DC predecessor cells) in vitro. Immature DCs actively take up antigens and pathogens, generate major histocompatability complex-peptide complexes, and migrate from the sites of antigen acquisition to secondary lymphoid organs to become mature dendritic cells that interact with and stimulate T-lymphocytes. During this process, the cells must undergo deformation to translocate through several barriers, including the basement membrane and interstitial connective tissue in the blood vessel wall. To further understand the mechanisms of the activation of immunological responses and the migration from peripheral tissue to secondary lymphoid organs, we have applied biophysical and microrheological methods to study the development processes of DCs in vitro. The results showed that membrane fluidity, osmotic fragility, membrane viscoelastic properties, infrared spectroscopy, and cytoskeleton organization of DCs exhibit significant differences in different developmental stages.

Studies on the biomechanical properties of maturing reticulocytes.

We investigated the biomechanical properties of reticulocytes obtained from an animal model of hemolytic anemia induced by antibody injection. The hemorheological indices, membrane viscoelasticity, membrane fluidity, and the secondary structure of membrane proteins of the reticulocytes were monitored continuously during the course of their maturation into erythrocytes. The results indicate that reticulocytes had lower deformability, lower membrane fluidity, greater viscoelastic modulus and lesser proportions of alpha-helices and beta-sheets in protein secondary structures than mature erythrocytes. All these indices approached to the level of normal erythrocytes when reticulocytes transformed during maturation. The results help to enhance our understanding of the biomechanical properties of the reticulocytes in their maturing process with clinical diagnosis significances.