Acidic microenvironment enhances MT1-MMP-mediated cancer cell motility through integrin ß1/cofilin/F-actin axis.

Tumor acidic microenvironment is the main feature of many solid tumors. As a part of the tumor microenvironment, it has a profound impact on the occurrence and development of tumors. However, the research on how tumor cells sense the changes of the external microenvironment and how the intracellular subcellular structures transmit the signals from extracellular to intracellular is unclear. In this study, we identify that the acidic microenvironment enhances cancer cell motility, and the expression of membrane-anchored membrane type 1-matrix metalloproteinase is also associated with cell motility, which indicates more degradation of the ECM under the acidic microenvironment. Moreover, the expression of cofilin is low in the acidic microenvironment, and the F-actin filaments are distributed more along the cells. The cytoskeletal F-actin changes are consistent with the potential of a high-invasive phenotype. Further study reveals the upstream control of the signal transductions from extracellular to intracellular, that is, the integrin ß1 functions to trigger the biological responses under the acidic microenvironment. Our results demonstrate that the acidic microenvironment enhances cancer cell motility through the integrin ß1/cofilin/F-actin signal axis. This study clearly shows the scheme of the signal transmissions from extracellular to intracellular and further reveals the cytoskeletal roles for the contributions of cancer cell motility under acidic microenvironment, which provides new targets for cancer intervention from the biochemical and biophysical perspectives.

Nitric oxide inhibits hypoxia-induced impairment of human RBC deformability through reducing the cross-linking of membrane protein band 3.

AIM: Nitric oxide (NO) prevents the decline of RBC deformability under high altitude and other ischemic and hypoxic conditions, but the clear mechanisms remain unknown. Here, we have carried out a systematic study to find the mechanisms of NO-induced regulation of RBC deformability under hypoxia. METHODS: NO levels, RBCs membrane elongation index (EI), membrane protein band 3 methemoglobin (MetHb) were determined during hypoxia (0 to 120 minutes). To validate the role of NO in regulating RBC deformability, tests were also performed with a NO donor (sodium nitroprusside) or a NO synthase inhibitor (l-nitro-arginine methylester) under 60 minutes hypoxia. RESULTS: Hypoxia for 45 minutes increased NO levels from 25.65 ± 1.95 to 35.26 ± 2.01 µmol/L, and there was a plateau after 60 minutes hypoxia. The EI did not change before 45 minutes hypoxia, but decreased from 0.567 ± 0.019 to 0.409 ± 0.042 (30 Pa) after 60 minutes hypoxia. The cross-linking of band 3 and phosphotyrosine increased after 45 minutes hypoxia. All can be alleviated by supplement NO and aggregated by inhibiting NOS. However, the MetHb was not present this trend. CONCLUSION: NO may prevent decreased of RBCs deformability through reducing the cross-linking of membrane band 3 under hypoxia; this helps microvascular perfusion of RBCs during ischemic and hypoxic disease states.

Exhaustive-exercise-induced oxidative stress alteration of erythrocyte oxygen release capacity.

The aim of the present study was to explore the effect of exhaustive running exercise in the oxygen release capacity of rat erythrocytes. Rats were divided into sedentary control, moderate running exercise, and exhaustive running exercise groups. The thermodynamic and kinetic properties of the erythrocyte oxygen release process of the different groups were tested. We also determined the degree of band-3 oxidation and phosphorylation, anion transport activity, and carbonic anhydrase isoform II activity. Biochemical studies suggested that exhaustive running significantly increased oxidative injury parameters in thiobarbituric acid reactive substances and methaemoglobin levels. Furthermore, exhaustive running significantly decreased anion transport activity and carbonic anhydrase isoform II activity. Thermodynamic analysis indicated that erythrocytes oxygen release ability also significantly increased due to elevated 2,3-DPG level after exhaustive running. Kinetic analysis indicated that exhaustive running resulted in significantly decreased T50 value. We presented evidence that exhaustive running remarkably impacted thermodynamic and kinetic properties of RBC oxygen release. In addition, changes in 2,3-DPG levels and band-3 oxidation and phosphorylation could be the driving force for exhaustive-running-induced alterations in erythrocyte oxygen release thermodynamic and kinetic properties.

Nitric oxide in red blood cell adaptation to hypoxia.

Nitric oxide (NO) appears to be involved in virtually every aspect of cardiovascular biology. Most attention has been focused on the role of endothelial-derived NO in basal blood flow regulation by relaxing vascular smooth muscle; however, it is now known that NO derived from red blood cells (RBCs) plays a fundamental role in vascular homeostasis by enhancing oxygen (O2) release at the cellular and physiological level. Hypoxia is an often seen problem in diverse conditions; systemic adaptations to hypoxia permit people to adjust to the hypoxic environment at high altitudes and to disease processes. In addition to the cardiopulmonary and hematologic adaptations that support systemic O2 delivery in hypoxia, RBCs assist through newly described NO-based mechanisms, in line with their vital role in O2 transport and delivery. Furthermore, to increase the local blood flow in proportion to metabolic demand, NO regulates membrane mechanical properties thereby modulating RBC deformability and O2 carrying-releasing function. In this review article, we focus on the effect of NO bioactivity on RBC-based mechanisms that regulate blood flow and RBC deformability. RBC adaptations to hypoxia are summarized, with particular attention to NO-dependent S-nitrosylation of membrane proteins and hemoglobin (S-nitrosohemoglobin). The NO/S-nitrosylation/RBC vasoregulatory cascade contributes fundamentally to the molecular understanding of the role of NO in human adaptation to hypoxia and may inform novel therapeutic strategies.

Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis.

BACKGROUND: In physiological and pathological conditions activated protein kinace C (PKC) has been observed in the erythrocytes. Externalization of ankyrin followed by Arg-Gly-Asp (RGD)/integrin recognition also triggers erythrophagocytosis. In the present study, to test whether activated PKC is associated with ankyrin exposure in erythrophagocytosis. METHODS: Phorbol 12-myristate-13-acetate (PMA)-induced PKC activation and ankyrin phosphorylation were tested, and under different treatment conditions the subpopulation of erythrocytes with ankyrin exposure and the levels of intracellular calcium were analyzed by flow cytometry. RESULTS: Results showed that treatment of erythrocytes with PMA in a calcium-containing buffer led to ankyrin exposure. In the absence of extracellular calcium, no ankyrin exposure was observed. PKC inhibition with calphostin C, a blocker of the PMA binding site, completely prevented the calcium entry, protein phosphorylation and ankyrin exposure. PKC inhibition with chelerythrine chloride, an inhibitor of the active site, diminished the level of ankyrin-exposing cells and ankyrin phosphorylation; however it even led to a higher percentage of cells with increased levels of calcium than with PMA treatment alone. Although PKC was activated and ankyrin phosphorylation occurred, no ankyrin exposure was observed in the absence of extracellular calcium. CONCLUSION: Analyses of results suggested that PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes and the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.

Exhaustive running exercise induce tyrosine phosphorylation of band 3 in rat erythrocytes.

BACKGROUND/AIMS: In vitro studies have shown that band-3 function is mainly regulated by its phosphorylation status. The main purpose of the study was to investigate whether band-3 phosphorylation status interferes with an exhaustive running exercise-related dysfunction of RBC deformability. METHODS: Rats were divided into sedentary control (C) and exercise test (ET) groups. The ET group was divided further into exhaustive running exercise (ERE) and moderate running exercise (MRE) subgroups. RESULTS: Tyrosine phosphorylation of band-3 was significantly elevated in the absence of reducing agent, consistent with the emergence of band-3 clustering in the ERE group compared with the control and MRE groups. The elongation index (EI) was found to decline significantly in the ERE group compared with the C and MRE groups under shear stress (control group, 0.41 ± 0.01 at 3 Pa and 0.571 ± 0.008 at 30 Pa; ERE group, 0.3140 ± 0.013 at 3 Pa and 0.534 ± 0.009 at 30 Pa; P < 0.001 and P < 0.002, respectively). CONCLUSION: Our results suggest that exhaustive running exercise results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization. Furthermore, it appears that exhaustive running exercise induced band 3 phosphorylation is due to the oxidation of critical sulfydryl groups of a membrane phosphatase (PTP).

Cluster of erythrocyte band 3: a potential molecular target of exhaustive exercise-induced dysfunction of erythrocyte deformability.

The aim of this study is to explore the effect of exhaustive exercise on erythrocyte band 3 (SLC4A1; EB3). The association between the alterations of EB3 and red blood cell (RBC) deformability induced by exercise-induced dysfunction has been investigated. Rats were divided among 2 groups: (i) control (C), and (ii) exercise exhausted (E). RBC deformability was investigated in the rats in the exhaustive exercise and control groups. Erythrocytes from the control and exercise-exhausted groups were evaluated for the expression of erythrocyte band 3 through immunoblotting and immunofluorescence studies. Exhaustive exercise led to significant increments in the levels of clustering of erythrocyte band 3 along with the conjugation of membrane proteins to form high-molecular-weight complexes (P < 0.05). Under shear stresses, RBC deformability was found to decline significantly in the exhaustive exercise groups compared with the control group. These data suggest that the RBC dysfunction observed during exercise-induced oxidative stress could be associated with alterations in the structure and function of erythrocyte band 3, which in turn leads to dysfunction in the rheological properties of RBCs. These results provide further insight into erythrocyte damage induced by exhaustive exercise.

Necroptosis in Organ Transplantation: Mechanisms and Potential Therapeutic Targets.

Organ transplantation remains the only treatment option for patients with end-stage organ dysfunction. However, there are numerous limitations that challenge its clinical application, including the shortage of organ donations, the quality of donated organs, injury during organ preservation and reperfusion, primary and chronic graft dysfunction, acute and chronic rejection, infection, and carcinogenesis in post-transplantation patients. Acute and chronic inflammation and cell death are two major underlying mechanisms for graft injury. Necroptosis is a type of programmed cell death involved in many diseases and has been studied in the setting of all major solid organ transplants, including the kidney, heart, liver, and lung. It is determined by the underlying donor organ conditions (e.g., age, alcohol consumption, fatty liver, hemorrhage shock, donation after circulatory death, etc.), preservation conditions and reperfusion, and allograft rejection. The specific molecular mechanisms of necroptosis have been uncovered in the organ transplantation setting, and potential targeting drugs have been identified. We hope this review article will promote more clinical research to determine the role of necroptosis and other types of programmed cell death in solid organ transplantation to alleviate the clinical burden of ischemia-reperfusion injury and graft rejection.

A pilot study on biological characteristics of human CD24(+) stem cells from the apical papilla.

BACKGROUND/PURPOSE: CD24 is a specific cell surface marker for undifferentiated dental stem cells from apical papilla (SCAPs) seen only during root development, before the tooth emerges through gum. But the comprehensive role of CD24 in the SCAPs is unclear. This study aims to clarify the exact roles of CD24 in SCAPs. MATERIALS AND METHODS: SCAPs were divided into CD24 (+)-SCAPs (high percentage CD24) and CD24 (-)-SCAPs (low percentage CD24) via flow cytometry. The proliferation, migration and osteogenic/adipogenic differentiation of the two groups were detected, RT-PCR was performed to detect the expression of osteogenic/adipogenic related genes and thegene expression were analyzed. RESULTS: The proliferative and migratory ability of CD24 (-)-SCAPs were significantly stronger than that of CD24 (+)-SCAPs. Although, the mineralization process and the osteogenic genes expression were not significantly difference in the two groups. Both CD24 (+)-SCAPs and CD24 (-)-SCAPs differentiated into adipocytes. The adipogenic differentiation in CD24 (+)-SCAPs was better than that in CD24 (-)-SCAPs, after 3 weeks of adipogenic induction. However, the expression of adipogenic related gene, PPAR γ2 mRNA in CD24 (+)-SCAPs was lower than that in CD24 (-)-SCAPs after 1 week of adipogenic induction. But the trend changed for the opposite after 3 weeks. CONCLUSION: The study proposes that CD24 has a regulatory effect on the adipogenic differentiation of SCAPs, and this may be attained by targeting the PPAR γ2 mRNA. Concurrently, it was found that CD24 plays an inhibitory role in the proliferation and migration of SCAPs, which may minimize the manifestation of diseases caused by an abnormal cell growth.

Simulation and Analysis of the Effects of Land Use and Climate Change on Carbon Dynamics in the Wuhan City Circle Area.

In a climate and land use change context, the sequestration of atmospheric carbon in urban agglomeration is key to achieving carbon emission and neutrality targets. It is thus critical to understand how various climate and land use changes impact overall carbon sequestration in large-scale city circle areas. As the largest urban agglomeration in central China, carbon dynamics in the Wuhan City Circle area have been deeply affected by rapid urbanization and climate change in the past two decades. Here, we applied monthly climate data, spatially explicit land use maps, NDVI (Normalized Difference Vegetation Index) images and the CASA (Carnegie-Ames-Stanford Approach) model to estimate the spatial and temporal changes of carbon dynamics in the Wuhan City Circle area from 2000 to 2015. We designed six different scenarios to analyze the effects of climate change and land use change on carbon dynamics. Our simulation of NPP (Net Primary Productivity) increased from 522.63 gC × m-2 to 615.82 gC × m-2 in the Wuhan City Circle area during 2000-2015. Climate change and land use change contributed to total carbon sequestration by -73.3 × 1010 gC and 480 × 1010 gC, respectively. Both precipitation and temperature had a negative effect on carbon sequestration, while radiation had a positive effect. In addition, the positive effect on carbon sequestration from afforestation was almost equal to the negative effect from urbanization between 2000 and 2015. Importantly, these findings highlight the possibility of carrying out both rapid urbanization and ecological restoration simultaneously.

Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis.

Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)-catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts.

Effect of curcumin extract against oxidative stress on both structure and deformation capability of red blood cell.

The normal deformability of erythrocytes plays an important role in ensuring blood mobility, erythrocyte longevity, and microcirculation, which is the ability of erythrocytes to change shapes in response to external forces. However, the effects of curcumin extracts on the deformability of erythrocytes have not yet been evaluated. Accordingly, in this study, we explored the effects of pre-treatment with curcumin extract on erythrocyte deformation and erythrocyte band 3 (SLC4A1; EB3) expression. We also evaluated the associations between EB3 expression and erythrocyte deformability induced by hydrogen peroxide. Blood samples were divided into the control group, pre-treatment group (treated with curcumin extract or vitamin C), and negative control group, and oxidant stress parameters, antioxidant status, erythrocyte deformability and elasticity, and EB3 modifications were evaluated using immunoblotting and immunofluorescence staining. Hydrogen peroxide significantly increased oxidative stress parameters, modulus elasticity values and clustered EB3 levels and induced conjugation of membrane proteins to form high-molecular-weight complexes (p < 0.05). Erythrocyte deformability and elasticity were significantly decreased in the treated groups compared with those in the control group. Overall, our findings suggested that pre-treatment with curcumin extracts increased antioxidant status, reduced EB3 cross-linking, and improved erythrocyte deformability, to an even better extent than vitamin C. These results provide important insights into the effects of treatment with curcumin extracts on erythrocyte damage and suggest that curcumin may have applications in antioxidant therapy.

Heparin as a molecular spacer immobilized on microspheres to improve blood compatibility in hemoperfusion.

Heparin, a highly sulfated linear polysaccharide, with anticoagulation function and blood compatibility is widely used as a biomaterials in medical application, but the most importance of heparin is its structure function as the macromolecular space arm. In this study, heparin as a spacer was covalently immobilized on the chloromethylated polystyrene microspheres (Ps) and then connected with l-phenylalanine forming the Ps-Hep-Phe structure, which was developed for endotoxin adsorption in hemoperfusion. The grafting density of heparin reach the maximum when the initial concentration of heparin solution was 5 mg/mL. The adsorbents with the heparin as a spacer showed the prolonged clotting times, low protein adsorption, and reduced the hemolysis rate, indicating that heparin-modified adsorbents have great blood compatibility. The adsorption capacity of Ps-Hep-Phe for endotoxin was 25.15 EU/g in dynamic adsorption, higher than that of Ps. Therefore, this study imply that heparin would be promising for modification of adsorbents in hemoperfusion.

[Effects of exhaustive exercise-induced oxidative stress on red blood cell deformability].

OBJECTIVE: The aim of the present study is to explore the effects of exhaustive exercise-induced oxidative stress on the antioxidant capacity and diformability of rat red blood cells. METHODS: Rats were divided into three group (n = 10): sedentary control (C), exhaustive running exercise (ERE) and moderate running exercise (MRE) groups. Animals in the ERE group started treadmill running at a speed of 20 m/min speed with a 5% gradient, and reached a speed of 25 m/min with gradient 15% in 20 min. Running was continued until exhaustion. MRE group rats running at a speed of 20 m/min with a 5% gradient for 40 min. The levels of free thiol in erythrocyte membrane protein, lipidperoxidation levels and membrane protein components were analyzed. The red blood cell deformability of different groups was also observed. RESULTS: The results showed that red blood cells were damaged by severe oxidative stress and the anti-oxidative capacity decreased significantly under exhaustive exercise conditions. Besides, lipid peroxidation and protein sulfhydryl cross-link based clustering of membrane were found after exhaustive exercise, and polymers high molecular weight (HMW) was formed. The elongation index (EI) was found to decline significantly in the ERE group compared with the C and MRE groups under shear stress (control group, 0.41 +/- 0.01 at 3 Pa and 0.571 +/- 0.008 at 30 Pa; ERE group, 0.314 +/- 0.013 at 3 Pa and 0.534 +/- 0.009 at 30 Pa; P < 0.05 and P < 0.01, respectively). CONCLUSION: These exercise-induced oxidative injure result in a significant decrease in deformability of rat erythrocytes, which in turn leads to dysfunction in the microcirculatory.