Optimization of the proportions of four wastewaters in a blend for the cultivation of microalgae using a mixture design.

A 2nd degree mixture design was used to determine the optimal blend prepared from four wastewater streams to produce microalgae-based biomass. The streams consisted of a liquid digestate from an anaerobic digestion process, a landfill leachate, a septic-system sludge treatment plant liquid, and a wastewater treatment plant effluent. The mixture regression analysis indicated that blends with higher proportions of treated effluent and digestate improved cells growth, while the use of leachate was detrimental to the growth. The global solution of the mixture optimization predicted a maximum value of biomass productivity of 22.76 mg L-1 d-1, in a blend consisting of 19% treated effluent, 21% digestate, and 60% water. Proportions of leachate higher than 13.33% were detrimental to the growth. The concentration of ammonia-N in the blends ranged from 0.39 to 150 mg L-1 d-1, and its toxicity effect on the cells diminished with increasing amounts of organic carbon in the cultivation medium.

Spontaneously hypertensive rats and platelet Ca(2+)-ATPases: specific up-regulation of the 97 kDa isoform.

The use of platelets instead of smooth muscle cells (SMC) to study the abnormal Ca2+ handling found in hypertension was investigated using spontaneously hypertensive rats (SHR). We studied the regulation of platelet Ca(2+)-ATPases, as we have recently demonstrated that human platelets, like SMC, contain the Ca(2+)-ATPase isoform termed SERCA2-b (sarco-endoplasmic reticulum Ca(2+)-ATPase). In mixed membranes isolated from platelets of normotensive Wistar-Kyoto (WKY) rats and SHR, total Ca(2+)-ATPase activity was found to be 43% higher in SHR than in WKY rats. By the use of autophosphorylation of rat platelet Ca(2+)-ATPases with [gamma-32P]ATP, followed by SDS/PAGE and Western blotting, we found that rat platelets express two distinct Ca(2+)-ATPases: a 100 kDa isoform, recognized by a SERCA2-b-specific anti-peptide antibody, and a 97 kDa isoform, specifically recognized by a polyclonal anti-SERCA antibody. Comparative analysis of platelet membrane Ca(2+)-ATPases from WKY rats and SHR demonstrated that the expression of the SERCA2-b isoform did not change significantly (128 +/- 22%), whereas that of the 97 kDa isoform reached 300 +/- 35% in SHR when compared with WKY rats. We concluded that the upregulation of total platelet Ca(2+)-ATPases in SHR is not due to the 100 kDa SERCA2-b isoform found in SMC, but is specific to the 97 kDa Ca(2+)-ATPase isoform which is not present in SMC. Therefore platelets should be used with extreme caution as a surrogate model of vascular smooth muscle Ca2+ homeostasis.

Performance of 45 laboratories participating in a proficiency testing program for Lyme disease serology.

OBJECTIVE: We show that significant interlaboratory and intralaboratory variations exist in Lyme disease proficiency testing. DESIGN: Six case-defined Lyme serum samples and three serum samples from individuals with no history of Lyme disease were randomized in four shipments and distributed to 45 participating laboratories. RESULTS: Interlaboratory and intralaboratory performances were highly variable. Approximately 4% to 21% of laboratories failed to identify correctly positive serum samples with titers of 512 or more using polyvalent serum or immunoglobulin G conjugates. With lower levels of anti-Borrelia burgdorferi antibody in the serum sample, approximately 55% of participating laboratories did not identify a case-defined serum. There was also a striking inability of many laboratories to reproduce their results on split samples from the same individual. In addition, 2% to 7% of laboratories identified serum samples from individuals with no known exposure to B burgdorferi as positive using polyvalent serum. The false positivity rate increased to 27% with the use of immunoglobulin G conjugate. CONCLUSIONS: Our results indicate that there is an urgent need for standardization of current testing methodologies. Until a national commitment is made, serological testing for Lyme disease will be of questionable value for the diagnosis of the disease.

The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2(+)-ATPase and is not glycoprotein Ib beta-subunit.

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

Further characterization of the plasma membrane- and intracellular membrane-associated platelet Ca2+ transport systems.

Biochemical characterization of the Ca2+-ATPases isolated from human platelet intracellular and plasma membranes is reported. A comparative study of the previously partly described plasma membrane Ca2+-ATPase [Enouf, Bredoux, Bourdeau & Levy-Toledano (1987) J. Biol. Chem. 261, 9293-9297] and the intracellular membrane Ca2+-ATPase obtained simultaneously shows differences in the following parameters: (1) different kinetics of the two enzymes; (2) similar apparent affinity towards Ca2+ (10(-7) M), though the intracellular membrane enzyme was inhibited at Ca2+ concentrations above 10(-6) M; (3) different pH dependence with an activity maximum at pH 7 for the intracellular membrane Ca2+-ATPase and no detectable pH maximum for the plasma membrane Ca2+-ATPase; (4) a 10-fold difference in the ATP requirement of the two Ca2+-ATPases; (5) different patterns of inhibition by vanadate. Finally, the possible regulation of the Ca2+-ATPases was examined by studying the effect of chlorpromazine on the two Ca2+-ATPase activities, with only the plasma membrane enzyme being inhibited. It is concluded that the two platelet Ca2+ transport systems show biochemical differences in spite of the previously shown similarity in the molecular masses of their Ca2+-ATPases, thus conferring a definite specificity to the platelet system.

Evidence of endoplasmic reticulum-related Ca2+ ATPase in human microvascular endothelial cells.

We have demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca2+-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca2+-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. 35S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca2+-ATPase competed. Dot-blotting experiments indicated that a substantial amount of Ca2+-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4-kb. As these experiments were conducted in comparison with cell types with well-defined Ca2+-ATPase in HOME cells.

Ajoene, the antiplatelet compound derived from garlic, specifically inhibits platelet release reaction by affecting the plasma membrane internal microviscosity.

Ajoene (E,Z-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a product of the rearrangement of allicin (a major component of raw garlic), has been shown to be a potent inhibitor of platelet aggregation in vitro through inhibition of granule release and fibrinogen binding. Our present study further elaborates on this inhibitory action, through studies of the effect of ajoene on the earliest steps of platelet activation. The transducing mechanism involved in thrombin-induced platelet activation was not modified by the drug as indicated by a normal breakdown of phosphatidylinositol 4,5,bisphosphate and normal production of phosphatidic acid. Likewise, the agonist-induced phosphorylation of myosin light chain (P20) and of the 43 kD protein (P43) were not impaired by ajoene. Under the same conditions, however, ajoene (100 microM) produced a strong inhibition of the thrombin-induced release of dense body and alpha-granule constituents. Electron spin resonance studies of the effect of ajoene on some physico-chemical properties of the platelet plasma membrane (intact platelets), as well as on artificial lipid membranes, indicated that ajoene increased mobility of the fatty acid spin label 16 nitroxide stearate. This suggests the existence of a decreased microviscosity of the most internal region within the lipid bilayer membrane, without affecting the outer hydrophilic moieties of the bilayer. As a whole, these results suggest that the effect of ajoene on the release reaction must be, in part, due to physical modification of the bilayer, which impairs the fusion of the granules and plasma membrane, a prerequisite for exocytosis.

Different sensitivity to trypsin of the human platelet plasma and intracellular membrane Ca2+ pumps.

The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.

Mechanisms of the mAb ALB6(CD9) induced human platelet activation: comparison with thrombin.

A CD9 monoclonal antibody described to aggregate human platelets was studied on different platelet functions in order to determine its mechanism of action. After a lag phase of 35 sec the mAb ALB6 induced a transient decrease in 32P-polyphosphoinositides, synthesis of 32P-phosphatidate (PA), phosphorylation of myosin light chain (P20) and of 43 KDa protein (P43) and the release reaction. Final biological and metabolic effects of ALB6 thus appear similar to that of thrombin but three differences bring additional information: (i) the lag phase, (ii) the kinetic of ALB6-induced release is identical for all granules whereas the release of dense granules is faster when induced by thrombin. (iii) no external Ca++ is required for ALB6 induced-activation.

Two different Ca2+ transport systems are associated with plasma and intracellular human platelet membranes.

Platelet plasma and intracellular membrane fractions were isolated from a mixed membrane fraction after sucrose cushion centrifugation. Their previous identification through biochemical and immunological characterization is now confirmed by sodium dodecyl sulfate electrophoresis of the membrane proteins which reveals a different protein profile. The two associated calcium transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate but the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes. We then focused on the study of the plasma membrane-associated Ca2+-activated ATPase which shows the following characteristics: a linearity in the time course until 30 min, an apparent affinity toward calcium of about 10(-7) M without detectable inhibition at higher concentrations, a maximal activity at pH 8, a high ATP requirement because the maximal response was obtained with 200 microM, and a high specificity toward ATP as energy donor. Taken together, these studies indicate the possible involvement of both a plasma membrane and a dense tubular system Ca2+-ATPase in the regulation of Ca2+ concentration in human platelets.

Stimulation of the 23-Kd protein cAMP dependent phosphorylation by inositol 1,4,5 trisphosphate in human platelet membrane vesicles.

The effect of inositol 1,4,5 trisphosphate (IP3) has been investigated on the cAMP-induced phosphorylation of the 23-Kd protein involved in platelet calcium fluxes by isolated membrane vesicles. The studies were conducted using the catalytic subunit of the cAMP-dependent protein kinase (C. Sub.). A dose-dependent stimulation of the 23 Kd protein phosphorylation induced by C. Sub. was initiated by IP3 with a half-maximal effect of 0.5 microM. The maximal effect was observed after 1-2 min. The effect was detected in the absence of Ca2+ and in the presence of phosphatase inhibitors. These results can suggest that the 23 Kd is an associated protein to the IP3 receptor in human platelets.

Relationship between cAMP and Ca2+ fluxes in human platelet membranes.

The effect of cAMP (which involved a 23 kDa protein phosphorylation) has been studied on the Ca2+ uptake and Ca2+ release from a human platelet membrane vesicle fraction. It was tested in the presence of the catalytic subunit of the cAMP-dependent protein kinase (C Sub). The addition of C Sub increased the steady state level of the Ca2+ uptake into the membrane vesicles. The effect was enhanced when tested in the absence of Ca2+ precipitating agent. The response was proportional to the dose of C Sub. Moreover, the effect varied with the Ca2+ concentration. The effect of C Sub has been tested on the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. A phosphorylated state of the 23 kDa protein appeared to be necessary. Indeed, a phosphorylation inhibition prevented the IP3 effect and the addition of C Sub increased the percentage of released Ca2+ (without modification of the time course). However, the C Sub dose-dependent response was not linear. The effect of cAMP on the two functions (Ca2+ uptake and Ca2+ release) appears to be different. Therefore, these results led us to suggest a more complex role of cAMP in the regulation of platelet Ca2+ concentration.

Possible role of a cAMP-dependent phosphorylation in the calcium release mediated by inositol 1,4,5-trisphosphate in human platelet membrane vesicles.

The addition of inositol 1,4,5-trisphosphate (IP3) to a 45Ca-preloaded human platelet membrane fraction (dense tubular system) induced a transient release of Ca2+. When the vesicle fraction was loaded with 45Ca2+ to isotopic equilibrium in the presence of the catalytic subunit of the cAMP-dependent protein kinase, the level of Ca2+ uptake was increased and the subsequent IP3-induced Ca2+ release was enhanced. The stimulation was observed regardless of the IP3 concentration used, and was maximal with an enzyme concentration of 5 micrograms/ml. The addition of the protein kinase inhibitor prevented the stimulatory effect of the catalytic subunit on IP3-induced calcium release, and also abolished the calcium release detected in the absence of added enzyme. It is concluded that a cAMP-dependent protein phosphorylation may be involved in the regulation of the IP3-induced Ca2+ release in human platelets.