An experimental model system for leishmaniasis. Effects of porphyrin-compounds and menadione on Leishmania parasites engulfed by cultured macrophages

In order to facilitate studies on the effects of chemotherapeutic agents on the host-parasite interactions in leishmaniasis, we have developed an experimental model for infecting human monocyte-derived- and mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigots (LDP). The chemotherapeutic agents studied were protoporphyrin, hematoporphyrin, menadione, and combinations of hematoporphyrin plus menadione. Since the Leishmania donovani amastigotes survived poorly in mouse macrophages and protoporphyrin was quite toxic to the latter, our investigations were focused on the effects of hematoporphyrin and menadione on amastigotes engulfed by human macrophages. Treatment of Leishmania donovani amastigotes-infested human macrophages with either 50 microM hematoporphyrin or 10 microM menadione did not influence significantly the survival of either Leishmania donovani amastigotes or the macrophages themselves. Larger individual doses of hematoporphyrin and menadione were toxic to both parasites and macrophages. The combination of 50 microM hematoporphyrin and 10 microM menadione, however, caused the destruction of the parasites without affecting the host macrophage. The enhanced deleterious effect from combined low doses of hematoporphyrin and menadione is discussed in terms of the production of reactive oxygen species, such as superoxide anion radical and hydrogen peroxide, originating from cellular redox cycling of menadione, and followed by decomposition of the formed hydrogen peroxide by hematoporphyrin to produce the more reactive hydroxyl radical.     

<Emphasis Type="Italic">In vivo</Emphasis> anti-inflammatory effect of a new steroidal saponin,mannioside A,and its derivatives isolated from <Emphasis Type="Italic">Dracaena mannii</Emphasis>

A new steroidal saponin, mannioside A (1), was isolated from the stem bark of Dracaena mannii, together with the known pennogenin (2), pennogenin-3-O-beta-D-glucopyranoside (3) and pennogenin-3-O-alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->3)]-beta-D-glucopyranoside (4). Their structures were determined using 1D-and 2D-NMR spectroscopy and mass spectrometry. Compounds 1 and 3 significantly inhibited carrageenan-induced paw edema in the rat; compound 4 was moderately active whereas 2 showed very weak activity.     

Dietary thioproline decreases spontaneous food intake and increases survival and neurological function in mice

Male mice on a diet supplemented with thioproline (l-thiazolidine-4-carboxylic acid), a physiological metabolite of 5-hydroxytryptamine, at 2.0 g/kg of food from 28 weeks of age and for their entire life, showed a 23-29% increased median and maximal life span. These survival increases were associated with improved neurological functions. Compared to control mice, thioproline-supplemented mice had a 20% lower integral spontaneous food intake, and 10% lower body weight at 100 weeks of age. Body weight showed a statistically significant inverse relationship with survival and neurological performances. Thioproline-supplemented mice exhibited a 58-70% decrease of the age-dependent oxidative damage in brain and liver mitochondria at 52 weeks (old mice) and 78 weeks (senescent mice) of age, respectively. The age-associated decrease of brain mitochondrial enzyme activities, NADH-dehydrogenase, cytochrome c oxidase, and mitochondrial nitric oxide synthase (mtNOS), in old and senescent mice were markedly prevented (51-74%) by thioproline. In vitro, thioproline neither exhibited direct antioxidant activity nor had any effect on the electron transfer or mtNOS functional activities of brain and liver mitochondria. It is surmised that thioproline induces an anorexic effect associated with improved survival and neurological function through a decreased oxidative damage and regulation that may involve hypothalamic appetite centers.     

Dysregulation of muscle fatty acid metabolism in type 2 diabetes is independent of malonyl-CoA

Aims/hypothesis An elevated lipid content within skeletal muscle cells is associated with the development of insulin resistance and type 2 diabetes mellitus. We hypothesised that in subjects with type 2 diabetes muscle malonyl-CoA (an inhibitor of fatty acid oxidation) would be elevated at baseline in comparison with control subjects and in particular during physiological hyperinsulinaemia with hyperglycaemia. Thus, fatty acids taken up by muscle would be shunted away from oxidation and towards storage (non-oxidative disposal).Materials and methods Six control subjects and six subjects with type 2 diabetes were studied after an overnight fast and during a hyperinsulinaemic (0.5 mU kg⁻¹ min⁻¹), hyperglycaemic clamp (with concurrent intralipid and heparin infusions) designed to increase muscle malonyl-CoA and inhibit fat oxidation. We used stable isotope methods, femoral arterial and venous catheterisation, and performed muscle biopsies to measure palmitate kinetics across the leg and muscle malonyl-CoA.Results Basal muscle malonyl-CoA concentrations were similar in control and type 2 diabetic subjects and increased (p<0.05) in both groups during the clamp (control, 0.14±0.05 to 0.24±0.05 pmol/mg; type 2 diabetes, 0.09±0.01 to 0.20±0.02 pmol/mg). Basal palmitate oxidation across the leg was not different between groups at baseline and decreased in both groups during the clamp (p<0.05). Palmitate uptake and non-oxidative disposal were significantly greater in the type 2 diabetic subjects at baseline and during the clamp (p<0.05).Conclusions/interpretation Contrary to our hypothesis, the dysregulation of muscle fatty acid metabolism in type 2 diabetes is independent of muscle malonyl-CoA. However, elevated fatty acid uptake in type 2 diabetes may be a key contributing factor to the increase in fatty acids being shunted towards storage within muscle.     

Cyclin D2 and cyclin D3 play opposite roles in mouse skin carcinogenesis

D-type cyclins are components of the cell-cycle engine that link cell signaling pathways and passage throughout G1 phase. We previously described the effects of overexpression cyclin D1, D2 or D3 in mouse epidermis and tumor development. We now asked whether cyclin D2 and/or cyclin D3 play a relevant role in ras-dependent tumorigenesis. Here, we described the effect of cyclin D3 and cyclin D2 overexpression in mouse skin tumor development. Notably, overexpression of cyclin D3 results in reduced tumor development and malignant progression to squamous cell carcinomas (SCC). Biochemical analysis of keratinocytes shows that overexpression of cyclin D3 results in strong reduction of cyclin D2 and its associated kinase activity. Furthermore, we found that reinstatement of cyclin D2 level in the cyclin D3/cyclin D2 bigenic mice results in a complete reversion of the inhibitory action of cyclin D3. Supporting these results, ablation of cyclin D2 results in reduced tumorigenesis and malignant progression. On the other hand, overexpression of cyclin D2 results in an increased number of papillomas and malignant progression. We conclude that cyclin D3 and cyclin D2 play opposite roles in mouse skin tumor development and that the suppressive activity of cyclin D3 is associated with cyclin D2 downregulation.     

Análisis psicométrico de las preguntas asociadas a imágenes radiológicas en el examen para médico interno residente en España

Background and objectivePsychometrics is a simple, intuitive approach used in educational research and in multiple-choice questionnaires. Since 2009, the competitive examination through which access to residency programs in Spain is determined (MIR) has included questions related to radiological images. The objective of this paper is to show the results of the psychometric analysis of these questions with the aim of comparing their degree of difficulty, discriminative capacity, and internal structure with respect to those of the other questions on the examination.Material and methodsWe analyzed all questions on the examination since 2009, classifying them as clinical cases with and without radiological images, clinical cases with and without non-radiological images, multiple choice questions, and negative questions. We used classical test theory and item response theory to assess the difficulty and degree of discrimination of the questions.ResultsOf 225 questions, between 11% and 15% of the questions included in the examinations were associated with images. The questions associated with radiological images were more difficult (corrected difficulty index, 0.51) and had worse discriminative capacity. The increased difficulty of radiological questions was associated with worse discriminative capacity, especially if the clinical information provided was inadequate or if the clinical information was contrary to the radiological concept or if there had never been any questions about the concept in previoous MIR examinations.ConclusionsTo equalize the standards of the MIR examination, it is necessary to maintain an appropriate structure in devising radiology questions, with terms from the clinical context, appropriate use of distracters, and a lower level of difficulty, which could be achieved by using radiological images with typical radiological findings.