Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

Alchemilla viridiflora Rothm.: the potent natural inhibitor of angiotensin I-converting enzyme

Molecular and Cellular Biochemistry - Alchemilla viridiflora Rothm., Rosaceae is a herbaceous plant widespread in central Greece, Bulgaria, North Macedonia and Serbia with Kosovo. Liquid...     

Teaching cardiovascular physiology with equivalent electronic circuits in a practically oriented teaching module

Here, we report on a new tool for teaching cardiovascular physiology and pathophysiology that promotes qualitative as well as quantitative thinking about time-dependent physiological phenomena. Quantification of steady and presteady-state (transient) cardiovascular phenomena is traditionally done by differential equations, but this is time consuming and unsuitable for most undergraduate medical students. As a result, quantitative thinking about time-dependent physiological phenomena is often not extensively dealt with in an undergraduate physiological course. However, basic concepts of steady and presteady state can be explained with relative simplicity, without the introduction of differential equation, with equivalent electronic circuits (EECs). We introduced undergraduate medical students to the concept of simulating cardiovascular phenomena with EECs. EEC simulations facilitate the understanding of simple or complex time-dependent cardiovascular physiological phenomena by stressing the analogies between EECs and physiological processes. Student perceptions on using EEC to simulate, study, and understand cardiovascular phenomena were documented over a 9-yr period, and the impact of the course on the students' knowledge of selected basic facts and concepts in cardiovascular physiology was evaluated over a 3-yr period. We conclude that EECs are a valuable tool for teaching cardiovascular physiology concepts and that EECs promote active learning.     

Phase variation: how to create and coordinate population diversity

Phase variation yields phenotypic heterogeneity in a clonal population as the result of one of a limited number of known molecular mechanisms. These include slipped strand mispairing, site-specific recombination and epigenetic regulation mediated by DNA methylation. Recently new regulatory variants utilizing these mechanisms have been identified, which is facilitating the identification of additional phase variation events solely from genome sequence analysis. Furthermore, it is becoming increasingly clear that in many cases phase variation control is integrated with regulatory networks and with cellular processes of a growing cell. This review focuses specifically on these recent advances in the understanding of the regulation of phase variation.     

A constitutive expression system for high‐throughput screening

To reduce the amount of consumables and number of pipetting steps in high‐throughput screening, a constitutive expression system was developed that comprises four different promoters of varying strength. The system was validated by the expression of different sucrose phosphorylase enzymes from Leuconostoc mesenteroides, Lactobacillus acidophilus and Bifidobacterium adolescentis in 96‐deep‐ and low‐well plates at three temperatures. Drastically improved soluble expression in mini‐cultures was observed for the enzymes from L. mesenteroides strains by reducing the promoter strength from strong to intermediate and by expressing the proteins at lower temperatures. In contrast, the enzymes from B. adolescentis and L. acidophilus were expressed most efficiently with a strong promoter. The constitutive expression of sucrose phosphorylases in low‐well plates resulted in a level of activity that is equal or even better than what was achieved by inducible expression. Therefore, our plasmid set with varying constitutive promoters will be an indispensable tool to optimize enzyme expression for high‐throughput screening.     

Progenitor cells in vascular disease

Stem cell research has the potential to provide solutions to many chronic diseases via the field of regeneration therapy. In vascular biology, endothelial progenitor cells (EPCs) have been identified as contributing to angiogenesis and hence have therapeutic potential to revascularise ischaemic tissues. EPCs have also been shown to endothelialise vascular grafts and therefore may contribute to endothelial maintenance. EPC number has been shown to be reduced in patients with cardiovascular disease, leading to speculation that atherosclerosis may be caused by a consumptive loss of endothelial repair capacity. Animal experiments have shown that EPCs reendothelialise injured vessels and that this reduces neointimal formation, confirming that EPCs have an atheroprotective effect. Smooth muscle cell accumulation in the neointimal space is characteristic of many forms of atherosclerosis, however the source of these cells is now thought to be from smooth muscle progenitor cells (SMPCs) rather than the adjacent media. There is evidence for the presence of SMPCs in the adventitia of animals and that SMPCs circulate in human blood. There is also data to support SMPCs contributing to neointimal formation but their origin remains unknown. This article will review the roles of EPCs and SMPCs in the development of vascular disease by examining experimental data from in vitro studies, animal models of atherosclerosis and clinical studies.     

Spatiotemporal allozyme variation in the damselfly, <Emphasis Type="BoldItalic">Lestes viridis</Emphasis> (Odonata: Zygoptera): gene flow among permanent and temporary ponds

Several insect species seem to persist not only in permanent but also in temporary ponds where they face particularly harsh conditions and frequent extinctions. Under such conditions, gene flow may prevent local adaptation to temporary ponds and may promote phenotypic plasticity, or maintain apparent population persistence. The few empirical studies on insects suggest the latter mechanism, but no studies so far quantified gene flow including both pond types. We investigated the effects of pond type and temporal variation on population genetic differentiation and gene flow in the damselfly Lestes viridis in northern Belgium. We report a survey of two allozyme loci (Gpi, Pgm) with polyacrylamide gel electrophoresis in 14 populations from permanent and temporary ponds, and compared these results with similar data from the same permanent populations one year before. The data suggested that neither pond-drying regime, nor temporal variation have a substantial effect on population genetic structuring and did not provide evidence for stable population differentiation in L. viridis in northern Belgium. Gene flow estimates were high within permanent and temporary ponds, and between pond types. Our data are consistent with a source-sink metapopulation system where temporary ponds act as sinks in dry years, and are quickly recolonized after local population extinction. This may create a pattern of apparent population persistence of this species in permanent and temporary ponds without clear local adaptation.     

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1^−/− mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1^−/− mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1^−/− mouse with ²H₇-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of ²H₇-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1^−/− mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.     

The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546–9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.