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.     

Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems

Eco R124I, Eco DXXI and Eco prrI are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while Eco prrI is chromosomally encoded. The enzymes are coded by three genes, hsdR , hsdM and hsdS . Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac , the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For Eco DXXI and Eco prrI the P1 homology extends for thousands of base pairs while for Eco R124I an IS 1 insertion and an associated deletion have removed most of the P1-homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.     

Retinoic acid synthesis by cytosol from the alcohol dehydrogenase negative deermouse

Cytosolic alcohol dehydrogenase in the deermouse is coded by a single genetic locus and a strain of the deermouse which is alcohol dehydrogenase negative exists. These two strains of the deermouse were used to extend insight into the role of cytosolic alcohol dehydrogenases in the conversion of retinol into retinoic acid. Retinoic acid synthesis from physiological concentrations of retinol (7.5 microM) with cytosol from the alcohol dehydrogenase negative deermouse was 13% (liver), 14% (kidney), 60% (testes), 78% (lung), and 100% (small intestinal mucosa) of that observed with cytosol from the positive deermouse. The rates in the negative strain ranged from 0.3 to 0.7 nmol/h/mg protein: sufficient to fulfill cellular needs for retinoic acid. Ten millimolar 4-methylpyrazole inhibited retinoic acid synthesis 92, 94, 26, and 30% in kidney, liver, lung, and testes of the positive deermouse, respectively, but only 50, 30, 0, and 0% in the same tissues from the negative deermouse. Ethanol (300 mM) did not inhibit retinoic acid synthesis in kidney cytosol from the negative strain. Therefore multiple cytosolic dehydrogenases, including alcohol dehydrogenases, contribute to retinol metabolism in vitro. The only enzyme(s) likely to be physiologically significant to retinoic acid synthesis in vivo, however, is the class of dehydrogenase, distinct from ethanol dehydrogenase, that is common to both the positive and the negative deermouse. This conclusion is supported by the data described above, the kinetics of retinoic acid synthesis and retinal reduction in kidney cytosol from the negative deermouse, and the very existence of the alcohol dehydrogenase negative deermouse. This work also shows that microsomes inhibit the cytosolic conversion of retinol into retinoic acid and that the synthesis of retinal, a retinoid that has no known function outside of the eye, does not reflect the ability or capacity of a sample to synthesize retinoic acid.     

Differential effect of cyclosporine A on respiratory burst by several types of human leukocytic cells.

The effect of cyclosporine on PMA-stimulated superoxide production has been studied on human alveolar macrophages, human neutrophils, cytoplasts and Epstein-Barr-virus-transformed B lymphocytes. Cyclosporine inhibits superoxide production in alveolar macrophages but not in neutrophils and cytoplasts. The respiratory burst of B-lymphocytes was scarcely inhibited by cyclosporine. The activity of NADPH oxidase from macrophages and neutrophils was not directly affected by cyclosporine. These data are considered in relation with the proposed mechanism for cyclosporine action and the stimulation of the respiratory burst.     

(23S)-1,23,25-Trihydroxycholecalciferol, an intestinal metabolite of 1,25-dihydroxycholecalciferol.

(23S)-23,25-Dihydroxycholecalciferol was converted into a polar metabolite in a calciferol-deficient chick kidney homogenate. The metabolite was identified as (23S)-1,23,25-trihydroxycholecalciferol by absorbance spectroscopy and mass spectrometry, and by formation of derivatives. (23S)-1,23,25-Trihydroxycholecalciferol was also observed as a 1,25-dihydroxycholecalciferol metabolite in intestinal cells isolated from 1,25-dihydroxycholecalciferol-treated rat. The trihydroxy metabolite was 50-fold less potent than 1,25-dihydroxycholecalciferol in the chick intestinal 1,25-dihydroxycholecalciferol receptor assay.     

High-grade mucoepidermoid carcinoma of the breast. Fine needle aspiration cytology and clinicopathologic study of a case

A primary high-grade mucoepidermoid carcinoma of the breast was evaluated preoperatively by fine needle aspiration (FNA) cytology in a 72-year-old woman. The smears showed a mixed pattern consisting of clusters of poorly differentiated squamous cells, keratinized squamous cells and vacuolated mucin-secreting cells. The subsequent mastectomy specimen showed a tumor with the features of a high-grade mucoepidermoid carcinoma. Electron microscopy confirmed the diagnosis, reflecting the epidermoid and glandular differentiation of the tumor. The course was rapidly fatal, and the patient died a few months after presentation. A review of the literature indicated that mucoepidermoid carcinoma of the breast is a very rare neoplasm; the FNA cytologic features described in this report may constitute a basis to preoperatively recognize this tumor.     

Molecular mechanisms of oxidative stress-related neonatal jaundice

Oxidative stress is a pathological condition characterized by an overload of oxidant products, named free radicals, which are not well counteracted by antioxidant systems. Free radicals induce oxidative damage to many body organs and systems. In neonatal red blood cells, free-radical mediated-oxidative stress leads to eryptosis, a suicidal death process of erythrocytes consequent to alteration of cell integrity. Neonatal red blood cells are targets and at the same time generators of free radicals through the Fenton and Haber-Weiss reactions. Enhanced eryptosis in case of oxidative stress damage may cause anemia if the increased loss of erythrocytes is not enough compensated by enhanced new erythrocytes synthesis. The oxidative disruption of the red cells may cause unconjugated idiopathic hyperbilirubinemia in neonates. High levels of bilirubin are recognized to be dangerous for the central nervous system in newborns, however, many studies have highlighted the antioxidant function of bilirubin. Recently, it has been suggested that physiologic concentration of bilirubin correlates with higher antioxidant status while high pathological bilirubin levels are associated with pro-oxidants effects. The aim of this educational review is to provide an updated understanding of the molecular mechanisms underlying erythrocyte oxidant injury and its reversal in neonatal idiopathic hyperbilirubinemia.