Mouse Red Blood Cells Research Articles

Methionine gamma-lyase-encapsulated into red blood cells (ERY-MET) antitumor activity in gastric carcinoma.

78 Background: Methionine (Met) requirement is a cancer specific–metabolic defect that seems a promising target, especially in gastric cancers. Methionine gamma–lyase (MGL), a pyridoxal–5′–phosphate (PLP)–dependent enzyme, is an emerging approach consisting in tumors Met starvation via systemic Met depletion. ERY-MET is a new therapeutic product overcoming the short in vivo half-life of free MGL by its encapsulation into Red Blood Cells (RBCs). Indeed, ERY-MET works as a bioreactor degrading Met that passively diffuses inside the RBC. In addition, entrapped MGL activity can be controlled by supplying Vitamin B6 (PN), the precursor of MGL’s cofactor (PLP), converted inside RBCs. ERY-MET anti-tumor activity was evaluated in vivo in NMRI nudemice bearing subcutaneous gastric carcinoma. Methods: First, in vitro sensitivity of NCI-N87 and AGS human gastric cell lines to free MGL was assessed by IC50 determination using CCK–8 assay. MGL encapsulated into mouse RBCs by hypotonic dialysis was injected once in CD1 mice to determine PK-PD parameters with or without PN supplementation. The anti-tumor activity of weekly ERY-MET injections (x5) at 116 IU/kg ± 25% was assessed with or without PN supplementation in female NMRI nudemice (n = 10/group) xenografted with NCI-N87 cells. Met depletion was determined 6 days after each cumulative injection while tumor growth was followed twice a week by caliper measurement. Results: In vitro studies showed that NCI-N87 as well as AGS cell lines displayed a sensitivity to free MGL with IC50 of 0.35 ± 0.01 and 0.12 ± 0.02 IU/mL, respectively. ERY-MET with daily PN supplementation significantly increased active MGL half-life in vivo (from < 24h to 8–9 days). ERY-MET induced 80% inhibition of tumor growth at day 45 (p < 0.0001). Response rate obtained was 76% of treated mice (15/20). Besides, PN supplementation induced a slow-down of tumor growth during the supplementation period and improved ERY-MET efficacy. Conclusions: Theses results suggest that ERY-MET can induce tumor growth inhibition in mice bearing human gastric adenocarcinoma and that its effect can be regulated by PN supplementation. As such, ERY-MET seems a promising anti-tumor drug to treat gastric cancers.

Does Erythropoietin Regulate TRPC Channels in Red Blood Cells?

Background: Cation channels play an essential role in red blood cells (RBCs) ion homeostasis. One set of ion channels are the transient receptor potential channels of canonical type (TRPC channels). The abundance of these channels in primary erythroblasts, erythroid cell lines and RBCs was associated with an increase in intracellular Ca²⁺ upon stimulation with Erythropoietin (Epo). In contrast two independent studies on Epo-treated patients revealed diminished basal Ca²⁺ concentration or reduced phosphatidylserine exposure to the outer membrane leaflet. Methods: To resolve the seemingly conflicting reports we challenged mature human and mouse RBCs of several genotypes with Epo and Prostaglandin E₂ (PGE₂) and recorded the intracellular Ca²⁺ content. Next Generation Sequencing was utilised to approach a molecular analysis of reticulocytes. Results/Conclusions: Our results allow concluding that Epo and PGE₂ regulation of the Ca²⁺ homeostasis is distinctly different between murine and human RBCs and that changes in intracellular Ca²⁺ upon Epo treatment is a primary rather than a compensatory effect. In human RBCs, Epo itself has no effect on Ca²⁺ fluxes but inhibits the PGE₂-induced Ca²⁺ entry. In murine mature RBCs functional evidence indicates TRPC4/C5 mediated Ca²⁺ entry activated by Epo whereas PGE₂ leads to a TRPC independent Ca²⁺ entry.

Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4.

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase-like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs.

Dematin and Adducin Tether Sodium-Hydrogen Exchanger, NHE1, to Erythrocyte Membrane Cytoskeleton

Dematin is a critical component of the membrane junctional complex in red blood cells. It tethers the spectrin cytoskeleton proteins to the membrane and its genetic deletion in mice causes dissociation of the spectrin, actin and β-adducin from the membrane resulting in the collapse of the red blood cells (RBCs). As dematin lacks a transmembrane domain, it is still unclear how this critical component of the junctional complex is anchored to the RBC membrane. Our previous studies have shown that the multi-transmembrane glucose transporter-1 (GLUT1) interacts with dematin and β-adducin in human RBCs, suggesting a potential role for GLUT1 in recruiting dematin to the membrane. However, as mouse RBCs do not express a GLUT1 homologue, an equivalent membrane receptor for dematin and/or adducin in mice remains to be determined. Using multiple in vitro and in vivo biochemical assays, here we demonstrate that the ubiquitously expressed plasma membrane Na+/H+ exchanger, NHE1 (Slc9a1), is one of the receptors for dematin and β-adducin in mature mouse red blood cells. NHE1 directly interacts with the core domain of dematin. Moreover, the dematin headpiece domain mutant S381E, which binds to the core domain with a higher affinity than the wild type, abolished the biochemical interaction between dematin and NHE1. This observation suggests that NHE1 and dematin headpiece domain compete for the same binding site(s) on the core domain. Furthermore, this finding highlights a molecular mechanism whereby an intermolecular switch of dematin regulates its interaction with NHE1 by phosphorylation. Dematin and β-adducin directly interact with NHE1 at its membrane-proximal cytoplasmic domain, which in turn regulates NHE1 activity in response to growth factor stimuli and intracellular pH alterations. Accordingly,we observed an increased cellular sodium content in erythrocytes of dematin headpiece and adducin double knockout mice (DAKO), suggesting a higher NHE1 activity in DAKO erythrocytes. Unlike GLUT1, NHE1 is expressed in both mouse and human RBCs. Thus, our results provide a novel mechanism for linking NHE1 to membrane skeleton and multiple cell signaling pathways through dematin and adducin (Figure 1). Since NHE1 is one of the major regulators of intracellular pH and hypertonic stress, our findings raise the possibility that the dematin-adducin-NHE1 complex may modulate these functions in RBCs as well as in other cell types with broad impact on the regulation of the actin cytoskeleton and cell migration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mitophagy Induction Is a Potential Therapeutic Approach for Sickle Cell Disease

▪Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. A single point mutation of the sixth amino acid of β-globin causes glutamic acid to be replaced by valine, rendering the hemoglobin susceptible to polymerization when deoxygenated. SCD patients suffer from the wide variety of disease manifestations including chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. In addition to the HbS polymerization-mediated rigid and fragile sickle-shaped red blood cell (RBC) formation, an excessive formation of intracellular reactive oxygen species (ROS) occurs in SCD red blood cells, which accelerates their hemolysis. This causes the release of ROS, free extracellular hemoglobin, hemin, and inflammatory cytokines that trigger disease progression. We analyzed levels of ROS in SCD patient RBCs and observed a higher fraction of intracellular ROS positive RBC in SCD (HbSS) compared to control (HbAA) RBC of adults [Control (HbAA): 7.1%± 1.4 %, n=11; SCD (HbSS): 25.3 % ± 4.3%, n=9; p<0.0004]. We also made the novel observation that mature RBCs from SCD patients abnormally contain mitochondria as evidenced by flow cytometry analysis of blood samples of 36 SCD patients and 14 normal human control subjects.[Control (HbAA):0.4 % ± 0.04%, n=14; SCD (HbSS): 7.8%± 0.9%, n=30; p<0.0001]. Further subset analysis from SCD patients with HbSC showed mitochondrial retention in their mature RBCs [HbSC: 2.2%± 0.6%,n=6 p<0.01], however to a lesser degree than patients with HbSS. Transmission electron microscopy confirmed the presence of mitochondria in mature RBC of patients with SCD. ROS analysis between mitochondria positive vs. negative fractions showed that mitochondria-positive (TMRM+) RBC fractions have higher levels of ROS compared to mitochondria-negative (TMRM-) RBC fractions. This data strongly suggests that retained mitochondria significantly contribute to the production of ROS in SCD RBCs. Similar to humans, a higher fraction of RBCs of SCD mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/J) retain mitochondria compared to control mice RBC [Control (HbAA): 0.29% ± 0.18%; SCD (HbSS): 16.68%± 1.9%, p<0.0001]. While investigating RN-1, a lysine specific demethylase-1 (LSD-1) inhibitor, as a HbF inducing agent, we observed that SCD mice treated with RN-1 showed a reduction in the fraction of RBCs which retain mitochondria. Therefore, we investigated mitophagy-inducing drugs as a possible useful therapeutic approach for SCD by administering mitophagy-inducing agent Sirolimus. SCD mice treated with RN-1 (5mg), or Sirolimus (5mg) had a significant decrease in the fraction of mitochondria containing RBCs (RN1: 4.96± 1.0%, p<0.0005; Sirolimus: 6.4% ± 1.8%, p<0.002). We observed a reduction of ROS in mature RBCs coupled with decreased mitochondrial retention in RBCs after in vivo treatment with RN1 or Sirolimus as measured by co-staining of TMRM, APC-conjugated CD71antibody, and CM-H2DCFDA. We also observed a significant improvement in RBC survival after the in vivo treatment with Sirolimus or RN-1. RBC survival was measured by flow cytometry and calculated biotin positive circulating RBCs after 2 days of in vivo labeling [SCD treated with vehicle control: 40 %± 2.6%; SCD treated with RN1 (2.5mg): 69.9 ± 2.6%, p<0.004; Sirolimus (5mg): 67.5% ± 6.1%, p<0.04]. Based on this data, mitophagy-inducing drugs have the potential to be a novel therapeutic approach for the treatment of SCD patients. DisclosuresJagadeeswaran:Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding. Rivers:Acetylon: Research Funding.

Antidiabetic studies of Chaetomorpha antennina extract using experimental models

Chaetomorpha antennina is a marine green alga found abundantly on the southern coast of India. This study explores the efficacy of C. antennina extracts in the management of diabetes using in vitro and in vivo models. The algal metabolites were extracted using various solvents and evaluated for inhibitory effect against key metabolic enzymes involved in blood sugar management and antioxidant property. Among the various extracts, methanolic extract of C. antennina (MECA) was found to be effective in all the inhibitory assays including, α-amylase (IC50 525.8 μg mL−1), α-glucosidase (IC50 121.3 μg mL−1), DPP-IV (IC50 24.92 μg mL−1) and antioxidant activity using DPPH (38 %) under in vitro conditions. In toxicity assays, MECA was nontoxic against mouse macrophage cells (J774) and red blood cells. Further, in vivo studies of MECA (250 mg kg−1 B.wt) on streptozotocin (STZ)-induced diabetic rats for a period of 28 days reduced the fasting blood glucose level to 39.97 % and that of positive control glibenclamide (0.25 mg kg−1 B.wt) was 73.05 % respectively. Serum cholesterol, triglycerides, ALT and AST were significantly decreased after 28 days of MECA treatment. GC-MS analysis of MECA showed the presence of ascorbic acid, octadecadienoate and fucosterol which were already identified for antidiabetic action. Overall, this study shows that the MECA exhibits antidiabetic activity in STZ-induced diabetic rats. Therefore, MECA can be considered for further studies to evaluate its mechanism for the effective management of diabetes.

The Nlrp3 Inflammasome Does Not Regulate Alloimmunization to Transfused Red Blood Cells in Mice

Red blood cell (RBC) transfusions are essential for patients with hematological disorders and bone marrow failure syndromes. Despite ABO matching, RBC transfusions can lead to production of alloantibodies against “minor” blood group antigens. Non-ABO alloimmunization is a leading cause of transfusion-associated mortality in the U.S. Despite its clinical importance, little is known about the immunological factors that promote alloimmunization. Prior studies indicate that inflammatory conditions place patients at higher risk for alloimmunization. Additionally, co-exposure to pro-inflammatory pathogen associated molecular patterns (PAMPs) promotes alloimmunization in animal models, suggesting that RBC alloimmunization depends on innate immune cell activation. However, the specific innate immune stimuli and sensors that induce a T cell-dependent alloantibody response to transfused RBCs have not been identified. The NLRP3 inflammasome senses chemically diverse PAMPs and damage associated molecular patterns (DAMPs), including extracellular ATP and iron-containing heme. We hypothesized that activation of the NLRP3 inflammasome by endogenous DAMPs from RBCs promotes the alloimmune response to a sterile RBC transfusion. Using genetically modified mice lacking either NLRP3 or multiple downstream inflammasome response elements, we ruled out a role for the NLRP3 inflammasome or any Caspase-1 or -11 dependent inflammasome in regulating RBC alloantibody production to a model antigen.

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.

The SOD1 transgene expressed in erythroid cells alleviates fatal phenotype in congenic NZB/NZW-F1 mice

Oxidative stress due to a superoxide dismutase 1 (SOD1) deficiency causes anemia and autoimmune responses, which are phenotypically similar to autoimmune hemolytic anemia (AIHA) and systemic lupus erythematosus (SLE) in C57BL/6 mice and aggravates AIHA pathogenesis in New Zealand black (NZB) mice. We report herein on an evaluation of the role of reactive oxygen species (ROS) in a model mouse with inherited SLE, that is, F1 mice of the NZB × New Zealand white (NZW) strain. The ROS levels within red blood cells (RBCs) of the F1 mice were similar to the NZW mice but lower compared to the NZB mice throughout adult period. Regarding SLE pathogenesis, we examined the effects of an SOD1 deficiency or the overexpression of human SOD1 in erythroid cells by establishing corresponding congenic F1 mice. A SOD1 deficiency caused an elevation in ROS production, methemoglobin content, and hyperoxidation of peroxiredoxin in RBC of the F1 mice, which were all consistent with elevated oxidative stress. However, while the overexpression of human SOD1 in erythroid cells extended the life span of the congenic F1 mice, the SOD1 deficiency had no effect on life span compared to wild-type F1 mice. It is generally recognized that NZW mice possess a larval defect in the immune system and that NZB mice trigger an autoimmune reaction in the F1 mice. Our results suggest that the oxidative insult originated from the NZB mouse background has a functional role in triggering an aberrant immune reaction, leading to fatal responses in F1 mice.

Loss of intrasplenic 33D1+DC migration impairs alloimmune responses against RBC transfusion

Abstract Although the migration of tissue DCs from periphery to draining LNs is the crucial step for the induction of T cell responses, it is not clear whether the same holds true within the spleen. Here we use red blood cell (RBC) transfusion model to specifically study intrasplenic DC migration in the induction of alloantibodies to blood group antigens present on the donor RBCs. We observed that mice deficient in DOCK8 (dedicator of cytokinesis 8), a guanine nucleotide exchange factor that regulates the activity of Rho GTPase family members, fail to produce alloantibodies following transfusion. Two major splenic conventional DC subsets, the Batf3-dependent CD8+ XCR1+ subset and the IRF-4-dependent CD11b+33D1+ subset, prime CD4+ and CD8+ T cells, respectively. Our data demonstrate that only bridging channel 33D1+ DCs migrate to the T cell zone to initiate CD4+ T cell responses during RBC transfusion. Using in vivo antibody labeling method, we are able to differentiate marginal zone/red pulp dendritic cells from T cell zone/white pulp dendritic cells. In vivo labeling reveals that the failure of generating alloantibodies to RBCs in DOCK8 deficient mice is due to impaired 33D1+ DC migration. Interestingly, although the XCR1+ DCs in DOCK8 deficient mice migrated normally, they played no role in alloimmune responses. Taken together, our data demonstrate that subset specific migration of DCs within spleen is critical for initiation of CD4 T cell response and generation of alloantibodies. As equivalent populations of DC subsets exist in humans, our work suggests that blocking certain DC subset migration therapeutically during RBC transfusion could potentially prevent the harmful sensitization that occurs in patients who require lifelong RBC support.

The Effect of Polymeric Nanoparticles on Biocompatibility of Carrier Red Blood Cells.

Red blood cells (RBCs) can be used for vascular delivery of encapsulated or surface-bound drugs and carriers. Coupling to RBC prolongs circulation of nanoparticles (NP, 200 nm spheres, a conventional model of polymeric drug delivery carrier) enabling their transfer to the pulmonary vasculature without provoking overt RBC elimination. However, little is known about more subtle and potentially harmful effects of drugs and drug carriers on RBCs. Here we devised high-throughput in vitro assays to determine the sensitivity of loaded RBCs to osmotic stress and other damaging insults that they may encounter in vivo (e.g. mechanical, oxidative and complement insults). Sensitivity of these tests is inversely proportional to RBC concentration in suspension and our results suggest that mouse RBCs are more sensitive to damaging factors than human RBCs. Loading RBCs by NP at 1:50 ratio did not affect RBCs, while 10–50 fold higher NP load accentuated RBC damage by mechanical, osmotic and oxidative stress. This extensive loading of RBC by NP also leads to RBCs agglutination in buffer; however, addition of albumin diminished this effect. These results provide a template for analyses of the effects of diverse cargoes loaded on carrier RBCs and indicate that: i) RBCs can tolerate carriage of NP at doses providing loading of millions of nanoparticles per microliter of blood; ii) tests using protein-free buffers and mouse RBCs may overestimate adversity that may be encountered in humans.

Calcium and Magnesium Ions Are Membrane-Active against Stationary-Phase Staphylococcus aureus with High Specificity.

Staphylococcus aureus (S. aureus) is notorious for its ability to acquire antibiotic-resistance, and antibiotic-resistant S. aureus has become a wide-spread cause of high mortality rate. Novel antimicrobials capable of eradicating S. aureus cells including antibiotic-resistant ones are thus highly desired. Membrane-active bactericides and species-specific antimicrobials are two promising sources of novel anti-infective agents for fighting against bacterial antibiotic-resistance. We herein show that Ca2+ and Mg2+, two alkaline-earth-metal ions physiologically essential for diverse living organisms, both disrupt model S. aureus membranes and kill stationary-phase S. aureus cells, indicative of membrane-activity. In contrast to S. aureus, Escherichia coli and Bacillus subtilis exhibit unaffected survival after similar treatment with these two cations, indicative of species-specific activity against S. aureus. Moreover, neither Ca2+ nor Mg2+ lyses mouse red blood cells, indicative of hemo-compatibility. This works suggests that Ca2+ and Mg2+ may have implications in targeted eradication of S. aureus pathogen including the antibiotic-resistant ones.

In vitro and In vivo Antioxidant Activity of Flavonoid Extracted from Mulberry Fruit (Morus alba L.).

Background:Many plants possess antioxidants that exhibit additive or synergistic activities.Objective:In this study, an ethanol-extracted flavonoid extracted from mulberry fruit (FEM) was evaluated for the antioxidant activity in vitro and the hemolysis in red blood cell (RBC) and lipid peroxidation in liver in vivo.Materials and Methods:Antioxidant activities in vitro were measured by quantifying its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power, and Fe2+-chelating ability. FEM inhibits hemolysis in RBCs and effects of lipid peroxidation in the liver were estimated.Results:The total content of flavonoid compounds was 187.23 mg of quercetin equivalents per grams dried material. In the in vitro assays, FEM demonstrated a strong antioxidant effect, especially in DPPH scavenging activity and reducing power. Mouse RBC hemolysis induced by H2O2 was significantly inhibited by FEM in a dose- and time-dependent manner. The effects of FEM on lipid peroxidation in liver, mitochondria, and microsome were investigated. The percentage of inhibition at high concentration (100 μg/mL) of FEM was 45.51%, 39.36%, and 42.78% for liver, mitochondria, and microsomes, respectively. These results suggest that the FEM possesses a strong antioxidant activity both in vivo and in vitro.SUMMARY The total content of flavonoid compounds in mulberry fruit was 187.23 mg/g dried materialFEM showed a strong antioxidant effect, especially in 2,2-diphenyl-1-picrylhydrazyl scavenging activity and reducing powerMouse red blood cell hemolysis induced by H2O2 was significantly inhibited by FEM in a dose- and time-dependent mannerThe inhibition percentage at high concentration of FEM was 45.51%, 39.36%, and 42.78% for mouse’s liver, mitochondrial, and microsomes, respectively. Abbreviations used: FEM: Flavonoid Extracted from Mulberry fruit, H2O2: Hydrogen peroxide, DPPH: 2,2-diphenyl-1-picrylhydrazyl, EDTA: Ethylene diamine tetraacetic acid, MDA: malondialdehyde, TBA: 2-thiobarbituric acid, RBC: Red blood cells, DNJ: 1-deoxynojirimycin, LDL: low density lipoprotein, ROS: reactive oxygen species, EDTA2Na: Ethylenediaminetetraacetic acid disodium salt.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Isolation of invasive Plasmodium yoelii merozoites with a long half-life to evaluate invasion dynamics and potential invasion inhibitors

Malaria symptoms and pathogenesis are caused by blood stage parasite burdens of Plasmodium spp., for which invasion of red blood cells (RBCs) by merozoites is essential. Successful targeting by either drugs or vaccines directed against the whole merozoite or its antigens during its transient extracellular status would contribute to malaria control by impeding RBC invasion. To understand merozoite invasion biology and mechanisms, it is desired to obtain merozoites that retain their invasion activity in vitro. Accordingly, methods have been developed to isolate invasive Plasmodium knowlesi and Plasmodium falciparum merozoites. Rodent malaria parasite models offer ease in laboratory maintenance and experimental genetic modifications; however, no methods have been reported regarding isolation of high numbers of invasive rodent malaria merozoites. In this study, Plasmodium yoelii-infected RBCs were obtained from infected mice, and mature schizont-infected RBCs enriched via Histodenz™ density gradients. Merozoites retaining invasion activity were then isolated by passing the preparations through a filter membrane. RBC-invaded parasites developed to mature stages in vitro in a synchronous manner. Isolated merozoites were evaluated for retention of invasion activity following storage at different temperatures prior to incubation with uninfected mouse RBCs. Isolated merozoites retained their invasion activity 4h after isolation at 10 or 15°C, whereas their invasion activity reduced to 0–10% within 30min when incubated on ice or at 37°C prior to RBC invasion assay. Images of merozoites at successive steps during RBC invasion were captured by light and transmission electron microscopy. Synthetic peptides derived from the amino acid sequence of the P. yoelii invasion protein RON2 efficiently inhibited RBC invasion. The developed method to isolate and keep invasive P. yoelii merozoites for up to 4h is a powerful tool to study the RBC invasion biology of this parasite. This method provides an important platform to evaluate the mode of action of drugs and vaccine candidates targeting the RBC invasion steps using rodent malaria model.

Low Red Blood Cell Vitamin C Concentrations Induce Red Blood Cell Fragility: A Link to Diabetes Via Glucose, Glucose Transporters, and Dehydroascorbic Acid

Strategies to prevent diabetic microvascular angiopathy focus on the vascular endothelium. Because red blood cells (RBCs) are less deformable in diabetes, we explored an original concept linking decreased RBC deformability to RBC ascorbate and hyperglycemia. We characterized ascorbate concentrations from human and mouse RBCs and plasma, and showed an inverse relationship between RBC ascorbate concentrations and deformability, measured by osmotic fragility. RBCs from ascorbate deficient mice were osmotically sensitive, appeared as spherocytes, and had decreased β-spectrin. These aberrancies reversed with ascorbate repletion in vivo. Under physiologic conditions, only ascorbate's oxidation product dehydroascorbic acid (DHA), a substrate for facilitated glucose transporters, was transported into mouse and human RBCs, with immediate intracellular reduction to ascorbate. In vitro, glucose inhibited entry of physiologic concentrations of dehydroascorbic acid into mouse and human RBCs. In vivo, plasma glucose concentrations in normal and diabetic mice and humans were inversely related to respective RBC ascorbate concentrations, as was osmotic fragility. Human RBC β-spectrin declined as diabetes worsened. Taken together, hyperglycemia in diabetes produced lower RBC ascorbate with increased RBC rigidity, a candidate to drive microvascular angiopathy. Because glucose transporter expression, DHA transport, and its inhibition by glucose differed for mouse versus human RBCs, human experimentation is indicated.

Channelizing the red blood cell: molecular biology competes with patch-clamp.

Channelizing the red blood cell: molecular biology competes with patch-clamp.

English

Lectins have been well studied and proved to play important roles in plant defense but information of legume lectins from non-legume plants has been rarely reported. A new legume lectin gene, designated as SmL 1, was cloned from Salvia miltiorrhiza Bunge, a famous traditional Chinese medicinal plant. The cDNA of SmL 1 was 919 bp in length and contained an 822 bp open reading frame (ORF) encoding a putative lectin precursor with two legume lectin domains. The deduced SML1 protein of SmL 1 shared 29 to 43% identities with other legume lectin sequences. Real time PCR analysis revealed that SmL 1 was predominantly expressed in the leaves and could be induced by pathogens and MeJA. The recombinant protein (rSmL1) of SmL 1 in Escherichia coli M15 was purified and showed agglutination activity towards rabbit and mouse red blood cells, and anti-bacterial activity against E. coli (ATCC35218), Pseudomonas lachrymans (PSL) and Xanthomonas campestris pv. Campestris (Pammel) Dowson (XC-1). Based on these results, SmL1 could play a role in medicinal plant disease control. Keywords: Anti-Bacterial Activity, Gene Expression, Legume Lectin, Recombinant Protein, Salvia miltiorrhiza Bunge

Dietary folic acid protects against genotoxicity in the red blood cells of mice

Folate is an essential B vitamin required for the de novo synthesis of purines, thymidylate and methionine. Folate deficiency can lead to mutations and genome instability, and has been shown to exacerbate the genotoxic potential of environmental toxins. We hypothesized that a folic acid (FA) deficient diet would induce genotoxicity in mice as measured by the Pig-a mutant phenotype (CD24-) and micronuclei (MN) in reticulocytes (RET) and red blood cells/normochromatic erythrocytes (RBC/NCE). Male Balb/c mice were fed a FA deficient (0mg/kg), control (2mg/kg) or supplemented (6mg/kg) diet from weaning for 18wk. Mice fed the deficient diet had 70% lower liver folate (p<0.001), 1.8 fold higher MN-RET (p<0.001), and 1.5 fold higher MN-NCE (p<0.001) than mice fed the control diet. RETCD24− and RBCCD24− frequencies were not different between mice fed the deficient and control diets. Compared to mice fed the FA supplemented diet, mice fed the deficient diet had 73% lower liver folate (p<0.001), a 2.0 fold increase in MN-RET (p<0.001), a 1.6 fold increase in MN-NCE (p<0.001) and 3.8 fold increase in RBCCD24− frequency (p=0.011). RETCD24− frequency did not differ between mice fed the deficient and supplemented diets. Our data suggest that FA adequacy protects against mutagenesis at the Pig-a locus and MN induction in the red blood cells of mice.

Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy.

Babesia microti causes “emergency” human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.