Distinct Combinations of Borrelia burgdorferi Sensu Lato Genospecies Found in Individual Questing Ticks from Europe

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.     

The effect of dietary antioxidants on lipofuscin accumulation in the crustacean brain

Lipofuscin accumulation is associated with ageing at the subcellular level. A strong correlation between lipofuscin and age has been found in crustaceans using histological techniques. This association has been proposed as the basis for a methodology to age crustaceans and in some cases lipofuscin levels were found to be better correlated with age than size. The experiment presented here was designed to test the potential effect of diet, in particular dietary antioxidants, on lipofuscin accumulation and age estimation.The shrimp, Penaeus japonicus, was reared in an aquaculture facility and fed commercial pellets with modified vitamins C and E contents. One group was fed with levels of vitamins C and E of 1000 and 150 mg/kg, respectively, and another group with 2500 and 5000 mg/kg, respectively. The experiment started when the shrimp were 19 weeks old. Samples were obtained at this point and at ages 33 and 43 weeks. Lipofuscin was measured in the nerve cords (antennal neuropils and oesophageal connectives) in an area adjacent to the brain.Dietary antioxidants significantly affected lipofuscin levels. High vitamin content in the diet resulted in lower percentage of the observed area covered with lipofuscin, lower lipofuscin granule density and lower average granule size. Gender had no effect on any of these variables and granule size did not significantly change within each treatment. Lipofuscin area and granule density increased with age in both vitamin treatments.These results suggest that age estimation using lipofuscin indices may be biased when: (1) wild populations are dispersed over diverse environments; (2) the age estimation of wild individuals is based on the results obtained using laboratory-reared individuals.     

Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.     

Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI₉₅ 0.70–0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.     

Study of the antidiabetic capacity of the VO(dmpp)2 complex

In this work we report biochemical ex vivo studies with a vanadium compound containing a pyridinone ligand, the bis(1,2-dimethyl-3-hydroxy-4-pyridinonate)oxovanadium (IV), V^IVO(dmpp)₂, which has shown to have promising antidiabetic activity. The experiments were carried out on primary adipocytes of 6-8 week old Wistar rats. Insulin-stimulated glucose uptake studies were performed using a radioactive assay by measuring the (U)-¹⁴C-glucose taken up by the isolated adipocytes for 30 min. Adipocytes were incubated with and without insulin and in the presence and absence of different concentrations of V^IVO(dmpp)₂ (100-500 μM) for 45 min. We observed that in a nontoxic concentration, as demonstrated by the Alamar Blue test, V^IVO(dmpp)₂ significantly increases glucose uptake, in the absence of insulin, by 5-folds higher than basal, and it has a significant inhibitory effect of 78% on free fatty acid release in isolated adipocytes from normal rats. We also demonstrated that it promotes the phosphorylation of Akt1, a key protein in the insulin signaling cascade. These results were compared with those obtained with another vanadium compound reported in the literature, with a similar structure, the bis(maltolato)oxovanadium (IV) (BMOV), which is now in clinical trials. Our ex vivo results clearly indicate that V^IVO(dmpp)₂ is a good candidate to be a promising drug for the treatment of diabetes and other metabolic disorders.     

Functional specialization and differential regulation of short‐chain carboxylic acid transporters in the pathogen Candida albicans

The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose‐poor microenvironments during infection and that the ability to use alternative non‐fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1‐GFP (green fluorescent protein) and Jen2‐GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1‐GFP fusion. In the murine model of systemic candidiasis approximately 20–25% of C. albicans cells infecting the kidney expressed Jen1‐GFP and Jen2‐GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose‐poor niches within the host, and that these short‐chain carboxylic acid transporters may be important in the early stages of infection.     

Differentiation of Human Umbilical Cord Matrix Mesenchymal Stem Cells into Neural-Like Progenitor Cells and Maturation into an Oligodendroglial-Like Lineage

Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton''s jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.     

Modification of the Starch Block Electrophoresis Method for the Preparation of Immunoglobulin A from Multiple Myeloma Sera on a Large Scale

Separation of immunoglobulin A from a large sample of human serum from patients with multiple myeloma was done by utilizing a large zone electrophoresis apparatus.