Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion

Glucose depletion of erythrocytes leads to activation of Ca²⁺-permeable cation channels, Ca²⁺ entry, activation of a Ca²⁺-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca²⁺ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKC, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 µM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 µM) and the phosphatase inhibitor okadaic acid (1–10 µM) mimicked the effect of glucose depletion and similarly led to translocation of PKC and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 µM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca²⁺ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion. cell volume; eryptosis; calcium; okadaic acid; staurosporine     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

Retinoic acid induced suicidal erythrocyte death.

Vitamin A and retinoic acid have previously been shown to confer some protection against a severe course of malaria by fostering the phagocytosis of parasitized erythrocytes. Phagocytosis of erythrocytes is stimulated by phosphatidylserine exposure at the cell surface. The present study has thus been performed to explore the effect of retinoic acid and the specific retinoic acid receptor (RAR) agonist 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB) on erythrocyte annexin V binding, which reflects phosphatidylserine exposure at the cell surface. A 24 hours exposure to either, retinoic acid (3 microM) or TTNPB (3 microM), indeed significantly increased annexin binding, an effect paralleled by decrease of forward scatter reflecting cell shrinkage. According to Fluo3 fluorescence, exposure to either, retinoic acid (10 microM, 24 hours) or TTNPB (10 microM, 6 hours), significantly increased cytosolic Ca(2+)-activity, a known trigger of phosphatidylserine exposure. Infection of erythrocytes with Plasmodium falciparum increased phosphatidylserine exposure, an effect increased in the presence of TTNPB. In conclusion, retinoid acid and TTNPB trigger phosphatididylserine exposure and cell shrinkage of erythrocytes, typical features of suicidal erythrocyte death or eryptosis. The eryptosis could participate in the accelerated clearance of parasitized erythrocytes from circulating blood following treatment with retinoids.     

Stimulation of suicidal erythrocyte death by methylglyoxal.

Diabetes increases the percentage of circulating erythrocytes exposing phosphatidylserine (PS) at the cell surface. PS-exposing erythrocytes are recognized, bound, engulfed and degraded by macrophages. Thus, PS exposure, a feature of suicidal erythrocyte death or eryptosis, accelerates clearance of affected erythrocytes from circulating blood. Moreover, PS-exposing erythrocytes bind to the vascular wall thus interfering with microcirculation. The present study explored mechanisms involved in the triggering of PS exposure by methylgloxal, an extra- and intracellular metabolite which is enhanced in diabetes. PS exposure, cell size and cytosolic Ca(2+)-activity after methylglyoxal treatment were measured by FACS analysis of annexin V binding, forward scatter and Fluo-3-fluorescence, respectively, and it was shown that the treatment significantly enhanced the percentage of PS-exposing erythrocytes at concentrations (0.3 microM) encountered in diabetic patients. Surprisingly, methylglyoxal did not significantly increase cytosolic Ca(2+) concentration, and at concentrations up to 3 microM, did not decrease the forward scatter. Instead, exposure to methylglyoxal inhibited glycolysis thus decreasing ATP and GSH concentrations. In conclusion, methylglyoxal impairs energy production and anti-oxidative defense, effects contributing to the enhanced PS exposure of circulating erythrocytes and eventually resulting in anemia and deranged microcirculation.     

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.     

A microbiological assay for sterigmatocystin,T-2 toxin and zearalenone

A survey was done to find microorganisms useful for assaying sterigmatocystin; T-2 toxin and zearalenone.Staphylococcus aureus was found to be sensitive to T-2 toxin and zearalenone;Bacillus cereus was found to be sensitive to T-2 toxin only; andEscherichia coli was sensitive to sterigmatocystin. The response of the organisms to sterigmatocystin; T-2 toxin and zearalenone was found to be linear between 4 and 100 μg with sterigmatocystin toE. coli; between 2 and 25 μg with T-2 toxin toStaph, aureus andB. cereus; and between 4 and 100 μg with zearalenone toStaph, aureus. The lower limits of sensitivity of the test were 2 μg T-2 toxin and zearalenone, and 4 μg sterigmatocystin. The assay is rapid (15–17 hrs); simple and inexpensive; and can be used to verify the toxicity of samples and to confirm thin layer chromatographic results.     

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37°C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37°C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.Open in a separate windowClick here to view.^{(75M, flv)}