Gel based silver staining of proteins is thought to occur by selective reduction of silver ions to insoluble metallic silver at specific initiation sites in the vicinity of the protein molecules. Silver stained protein bands generally are dark brown or black with considerable variation in color intensity. The color variation has been attributed to diffractive scattering by silver grains of different sizes. Our experiments, however, demonstrate that color variation is due to the formation of silver chromate deposits that are incorporated into formalin fixed proteins. Understanding the mechanism of silver staining is essential for developing a method for protein quantification.

The components of the blood stain, eosin and methylene blue, were introduced by Baeyer and Caro, respectively. Methylene blue was used primarily for detecting Mycobacterium tuberculosis until Ehrlich in 1880 mixed methylene blue with acid fuchsin to produce what he termed a "neutral stain," which allowed differentiation of blood cells. Eight years later, Chęciń ski changed the acidic component of the dye to eosin. Plehn subsequently altered the proportions of eosin and methylene blue to produce a greater range of red and blue hues. In 1891, Malachowski and Romanowsky independently developed stains composed of eosin and "ripened" methylene blue that not only differentiated blood cells, but also demonstrated the nuclei of malarial parasites. A number of "ripening" or "polychroming" techniques were investigated by different groups, but the aqueous dye solutions produced were unstable and precipitated rapidly. Subsequently, methanol was introduced as a solvent for the dye precipitate and techniques were developed that utilized the fixative properties of the methanolic solution prior to aqueous dilution for staining. This avoided the troublesome process of heat fixation of blood films. Giemsa further improved these techniques by using more controlled methods of methylene blue demethylation. In addition, he used measured amounts of known dyes and increased dye stability by adding glycerol to the methanol solvent. With the outbreak of World War I, it became difficult to obtain German dyes outside of Germany; during the World War II, it became impossible. In their effort to improve the inferior American versions of Giemsa's stain, Lillie, Roe, and Wilcox discovered that the best staining results were obtained using pure methylene blue, one of its breakdown products (azure B) and eosin. These three substituents remain the major components of the stain to this day.

The biochemical pathway for the production of malaria pigment (haemozoin) forms a fundamental difference between host and parasite and is likely to be an important drug target. A simple method for the isolation of malaria pigment is described. The resultant product retained the in vivo crystalline appearance of pigment as judged by polarizing microscopy. Conditions were found for the disaggregation and separation of pigment. As malaria pigment can adsorb drugs, haem, and iron, such separation techniques are useful tools for studies on the impairment of haemoglobin digestion and pigment formation by antimalarials.

Experimental work was carried out to establish the growth characteristics of Plasmodium falciparum in an in vitro culture system using cells with the Dantu, Henshaw and S-s-U- blood-group variants. A flow cytometric technique, using the dye thiazole orange, was adapted for use on the Epics Profile II flow cytometer to count the parasites. This was performed at 24, 48 and 72 h. The ability of the parasites to grow in red cells of the Dantu and Henshaw phenotypes was also assayed by 3[H] hypoxanthine incorporation. Relative to control red cells, S-s-U- cells and Dantu cells were less suitable as host cells for P. falciparum in vitro. In contrast, cells expressing the Henshaw antigen were equally sensitive to P. falciparum infection as were normal controls. These data support the notion that glycophorins play an important role in P. falciparum infection. Further studies are required to evaluate the epidemiological significance of these results.

Extracellular iron is necessary for many biochemical reactions involved in Plasmodium falciparum growth and multiplication. The incorporation of radioactive iron taken up by the parasite was found, electrophoretically and via gamma counting, to be mainly associated with the haemozoin only in the presence of the active metabolism of the parasite. The potent antimalarial activity of desferrioxamine, a ferric iron chelating agent, has shown that iron deprivation is inhibitory to the parasite. We propose that the mechanism of action of desferrioxamine in addition to the chelation of iron from the parasitic compartment, chelates iron from the haemozoin crystal resulting in free radical generation and parasite death. The ability of desferrioxamine and not the ferrous iron chelating agent, 2,2'-bipyridyl, to chelate the non-haem iron from the haemozoin structure indicates that the oxidative state of iron associated with the haemozoin structure is ferric in nature.

Oxidative stress may affect the malaria parasite at each stage of its development. Ascorbic acid had a stage-dependent effect on the in vitro development of Plasmodium falciparum. Addition of ascorbic acid to the early stage (ring forms) increased hypoxanthine incorporation but prevented the development of trophozoites to mature forms of the plasmodium. We also used DNA degradation to monitor the levels of redox-active iron-containing structures that can catalyse or promote the production of free radicals. We found that DNA was degraded upon incubation with lysates of infected red blood cells. Ascorbic acid was found to enhance the efficiency of degradation whereas the chelating agent diethylnetriaminepentaacetic acid inhibited the effect.

Malarial parasites are believed to be more susceptible to oxidative stress than their hosts. BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) and HeCNU(1-(2-chloroethyl)-3-(2-hydroxythyl)-1-nitrosourea), inhibitors of the antioxidant enzyme glutathione reductase, were found to prevent the growth of Plasmodium falciparum in all intraerythrocytic stages. When exposing infected red blood cells to 38 microM BCNU or 62 microM HeCNU for one life cycle of synchronously growing parasites, the parasitemia decreased by 90%. During the formation of new ring forms, the parasites are even more susceptible to these drugs. The treatment with BCNU or HeCNU produced a rapid depletion of GSH in the parasites and their host cells; in addition, protection against lipid peroxidation was impaired in these cells. Possible mechanisms for the antimalarial action of the inhibitors are discussed. Our results suggest that erythrocyte glutathione reductase, an enzyme of known structure, might be considered as a target for the design of antimalarial drugs.

An improved protein-blotting procedure and a thin layer isoelectric focusing technique are introduced to study glutathione reductase and methemoglobin (Met-Hb). According to our results, there is only one form of glutathione reductase in normal red blood cells. A similar protein was shown to be present at higher concentration in isolated merozoites. Both proteins have a subunit Mr of ca. 50,000 and react with anti-human glutathione reductase serum. Red cells with schizonts do not possess a higher proportion of Met-Hb than non-parasitized erythrocytes. This finding suggests that Met-Hb is not an indicator of metabolic alterations in malaria-infected erythrocytes.

The glutathione metabolism of Plasmodium falciparum, P. vinckei and P. berghei has been investigated. Human erythrocytes with low glutathione reductase and synthetase activity are still capable of harbouring P. falciparum. Both enzymes have been demonstrated in Plasmodium spp. Moreover, evidence is given for a selenium-independent glutathione peroxidase in malaria parasites.

Ferriprotoporphyrin IX(FP) lysed both erythrocytes and isolated Plasmodium falciparum as judged by decrease in turbidity of erythrocyte and parasite suspensions. The lytic effect of FP on erythrocytes was enhanced by chloroquine(CQ). In the presence of 2.5–20 μM CQ, 5 μM FP led to complete hemolysis within 45 min. However, the lytic effect of FP or FP-CQ on both erythrocytes and parasites was inhibited completely by proteins. The protein inhibition was non-specific.

SUMMARYElectrophoresis of extracts of schizonts ofPlasmodium knowlesiin non-dissociating polyacrylamide gels, separates several bands of acid endopeptidase activity. A polyclonal antiserum, produced by immunization with purified merozoites, failed to distinguish between different bands of the parasite enzyme, indicating that they are serologically related. Apart from the loss of one minor peak, extraction in Triton X-100 did not reduce the enzyme's electrophoretic heterogeneity. The antiserum did not react with red cell acid proteases.

SUMMARY Polyclonal antisera raised against Plasmodium knowlesi reacted with (1) NADP-specific glutamate dehydrogenase (GLDH) of P. knowlesi, (2) GLDH of P. falciparum and (3) GLDH of Proteus spp. The antisera did not react with NAD(P) GLDH from bovine liver. Polyclonal antisera raised against the GLDH of Proteus spp. cross-reacted with GLDH from P. falciparum. Monoclonal antibodies (McAbs) obtained from mice immunized with Proteus GLDH were either specific for the bacterial enzyme or cross-reacted with P. falciparum GLDH. The selected McAbs did not react with GLDI-1 from P. knowlesi, P. chabaudi or P. berghei. The GLDH of P. falciparum was shown to be a cytosolic protein (by FAT) with a subunit molecular weight of approximately 49000 Da (by immunoprecipitation) having a pre dominantly hexameric form (by sucrose density gradient). Implications of the conserved sequences of GLDHs and other enzymes are discussed.

ABSTRACTThe proteins of homogenized adultOpisthorchis viverriniwere separated by polyacrylamide gel electro-Phoresis and isoelectric focusing in the presence of detergents. By far the most abundant component(s) had a Mr of 18,000 to 19,000, represented about 47% of the total parasite protein, and did not separate in isotypes.