A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.     

Fluorimetric determination of carbamoyl phosphate

A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP⁺, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present.     

Stabilization of ornithine decarboxylase in erythroleukemia cells depleted of ATP

Ornithine decarboxylase activity in Friend erythroleukemia cells decayed with a half-life of 50 minutes after addition of cycloheximide and at a faster rate after addition of spermidine. Incubation with a medium containing dinitrophenol and 2-deoxy-glucose in place of glucose caused ATP depletion and blocked the turnover of ornithine decarboxylase, even after addition of spermidine. Dinitrophenol in the presence of glucose was able to provoke only a slight increase of the half-life of the enzyme. These results suggest that degradation of ornithine decarboxylase in erythroleukemia cells is ATP-dependent.     

Polyamines stimulate superoxide production in human neutrophils activated by N-fMet-Leu-Phe but not by phorbol myristate acetate

The polyamines putrescine, spermidine and spermine, at concentrations of 10 microM, stimulated superoxide generation by human polymorphonuclear leukocytes induced by fMet-Leu-Phe in the presence of Ca2+. This positive effect was not evident in the absence of Ca2+ or when the polymorphonuclear leukocytes were stimulated by phorbol myristate acetate. Spermidine in the range of 10-100 microM showed a dose-dependent stimulatory effect on the superoxide generation induced by fMet-Leu-Phe, whilst at doses above 25 mM it produced an inhibitory effect. At this concentration, spermidine did not reduce the phorbol myristate acetate-neutrophil-induced O2-. generation, while an inhibitory effect by the polyamine was evident at concentrations above 50 mM. In addition, 100 microM spermidine increased the amount of superoxide generated and enhanced the ability of the chemotactic peptide to stimulate superoxide generation. The polyamines in the range of 10 microM-25 mM did not modify the activity of purified NADPH oxidase, nor the rate of reduction of cytochrome c as supported by the xanthine/xanthine oxidase reaction. These results indicate that physiological concentrations of polyamines can stimulate superoxide formation by polymorphonuclear leukocyte cells produced by the chemotactic peptide fMet-Leu-Phe, probably by increasing the availability of external calcium.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

Accumulation and Interconversion of Amino Acids in Rice Roots under Anoxia

In excised rice roots, anaerobic degradation of proteins gaverise to an increase of free-amino acids. Anoxic accumulationof alanine, -aminobutyric acid and proline was the consequenceof the interconversion of glutamate, aspartate and amides. Theshift in the composition of the amino acid pool appears to becaused by changes in keto acid levels. The role of reactionsinvolved in amino acid interconversion and the physiologicalsignificance of these interconversions are considered and discussed. (Received November 25, 1988; Accepted June 9, 1988)     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.