Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.     

Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity

We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.     

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.     

Compartmentalization of Ca2+ in sickle cells

Control (AA) and sickle cell anemia (SS) erythrocytes were loaded with Ca-chelator (Quin2 or Benz2) to increase the cellular exchangeable Ca2+ pool and to measure the Ca2+ exchange fluxes and the cytosolic ionized Ca2+ ([Ca]i) (Lew et al., 1982, Nature, 298, 478). The chelator incorporation induced a decrease in the ATP content which was smaller in SS than in AA cells and partially reversible upon reincubation in a chelator-free medium. The amount of trapped chelator was determined by two methods: 45Ca binding to the chelator in Ca-ionophore treated cells in Ca-EGTA buffers and [3H]Quin2 incorporation. A slight over-estimation of the chelator content was found with the second method but incorporation was the same in both types of cells. The kinetics of 45Ca equilibration and 45Ca release were used to measure Ca2+ fluxes and [Ca]i in oxygenated chelator-loaded cells. SS cells, as compared to AA cells, exhibited a moderate increase in Ca2+ fluxes (30-75%) but [Ca]i remained in the same range (about 20 nM). Thus the excess of Ca2+ found in SS cells is not available for the Ca2+ pump or the K+ channel a conclusion in agreement with that of Bookchin et al. (1984, Cell Calcium, 5, 277). Analysis of the 45Ca kinetics showed that in AA cells, exchangeable Ca2+ behaved as one compartment. In SS cells, the existence of a second slowly-exchangeable Ca2+ compartment was demonstrated. This latter (3-5 mumol/l cells) was independent of the concentration of the chelator and thus could represent exchangeable Ca2+ enclosed within the intracellular inside-out vesicles recently observed in SS cells (Williamson et al., 1984, J. Cell. Biol., 99, 430a). Alternatively, these two kinetic pools could reflect heterogeneity of the SS cell population.     

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells

Na⁺-ATPase of high-K⁺ and low-K⁺ sheep red cells was examined with respect to the sidedness of Na⁺ and K⁺ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na⁺ in the medium, i.e., at the cytoplasmic surface, Na_cyt⁺, the activation curves show that high-K⁺ Na⁺-ATPase has a higher affinity for Na_cyt⁺ compared to low-K⁺. The apparent affinity for Na_cyt⁺ is also increased by increasing the ATP concentrations in high-K⁺ but not low-K⁺. With Na_cyt⁺ present, Na⁺-ATPase is stimulated by intravesicular Na⁺, i.e., Na⁺ at the originally external surface, Na_ext⁺, to a greater extent in low-K⁺ than high-K⁺. Intravesicular K⁺ (K_ext⁺) activates Na⁺-ATPase in high-K⁺ but not in low-K⁺ vesicles and extravesicular K⁺ (K_cyt⁺) inhibits low-K⁺ but not high-K⁺ Na⁺-ATPase. Thus, the genetic difference between high-K⁺ and low-K⁺ is expressed as differences in apparent affinities for both Na⁺ and K⁺ and these differences are evident at both cytoplasmic and external membrane surfaces.     

Mouse macrophage elastase. Purification and characterization as a metalloproteinase.

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.     

Glutathione-related inhibition of prostaglandin metabolism

Placental homogenates contain a heat-stable, dialyzable fraction which specifically inhibits two placental enzymes, both of which possess 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The inhibition of the two enzymes is the same. The inhibitor has been resolved into two components by gel filtration on a column of Sephadex LH-20. The component which eluted first has been identified as oxidized glutathione (GSSG), the other as a glutathione-containing material (GSX). Inhibition of the 15-hydroxyprostaglandin dehydrogenase activity is competitive with respect to the prostaglandin substrate (K_i^GSSG = 26 μM, K_i^GSX = 1.4 μM). Inhibition of the 9-ketoprostaglandin reductase activity is also competitive with respect to the prostaglandin substrate (K_i^GSSG = 68 μM). The most effective inhibitor of the 15-hydroxyprostaglandin dehydrogenase is the prostaglandin A₁-glutathione adduct (K_i = 0.27 μM). This compound is not a substrate for oxidation of the 15-hydroxyl group but it is the best substrate found to date for reduction of the 9-keto function.     

Active potassium transport in reticulocytes of high-K and low-K sheep

The kinetics of active K⁺ transport were studied in immature red blood cells cells from high-K⁺ and low-K⁺ sheep, particularly with respect to the effects of varying intracellular K⁺ concentration, [K]_i. Comparison was made with active transport, or pump, activity in mature high-K⁺ and low-K⁺ red cells. Reticulocytes from both types of sheep had much higher maximal active K⁺ influxes than did mature cells. In both types of reticulocytes, and in mature high-K⁺ cells as well, the pump was relatively insensitive to increasing [K]_i. In contrast, intracellular K⁺ markedly inhibited the pump in mature low-K⁺ cells. Active K⁺ transport in low-K⁺ reticulocytes, however, as in mature low-K⁺ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K⁺ pumps of high-K⁺ and low-K⁺ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K⁺ and low-K⁺ cells with K⁺ pumps of very different kinetic characteristics.     

Synthesis and biological evaluation of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives as potential anticancer agents

A series of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives 5, 6 and 7 were prepared starting from 6-phenyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 3 via selective N-alkylation, followed by reaction with different primary aliphatic amines, cyclic secondary amines or l-amino acids under different set of conditions. All the synthesized compounds 5, 6 and 7 were screened for anticancer activity against four cancer cell lines such as A549—Lung cancer (CCL-185), MCF7—Breast cancer (HTB-22), DU145—Prostate cancer (HTB-81) and HeLa—Cervical cancer (CCL-2). The compounds 5i and 6e are found to have promising bioactivity at micro molar concentration.     

Strengthening Community-Based Vital Events Reporting for Real-Time Monitoring of Under-Five Mortality: Lessons Learned from the Balaka and Salima Districts in Malawi

Background

Malawi ratified a compulsory birth and death registration system in 2009. Until it captures complete coverage of vital events, Malawi relies on other data sources to calculate mortality estimates. We tested a community-based method to estimate annual under-five mortality rates (U5MR) through the Real-Time Monitoring of Under-Five Mortality (RMM) project in Malawi. We implemented RMM in two phases, and conducted an independent evaluation of phase one after 21 months of implementation. We present results of the phase two validation that covers the full project time span, and compare the results to those of the phase one validation.

Methods and Findings

We assessed the completeness of the counts of births and deaths and the accuracy of disaggregated U5MR from the community-based method against a retrospective full pregnancy history for rolling twelve-month periods after the independent evaluation. We used full pregnancy histories collected through household interviews carried out between November 2013 and January 2014 as the validation data source. Health Surveillance Agents (HSAs) across the 160 catchment areas submitted routine reports on pregnancies, births, and deaths consistently. However, for the 15-month implementation period post-evaluation, average completeness of birth event reporting was 76%, whereas average completeness of death event reporting was 67% relative to that expected from a comparable pregnancy history. HSAs underestimated the U5MR by an average of 21% relative to that estimated from a comparable pregnancy history.

Conclusions

On a medium scale, the community-based RMM method in Malawi produced substantial underestimates of annualized U5MR relative to those obtained from a full pregnancy history, despite the additional incentives and quality-control activities. We were not able to achieve an optimum level of incentive and support to make the system work while ensuring sustainability. Lessons learned from the implementation of RMM can inform programs supporting community-based interventions through HSAs in Malawi.