Polyisoprenoid epoxides stimulate the biosynthesis of coenzyme Q and inhibit cholesterol synthesis

In our search for compounds that up-regulate the biosynthesis of coenzyme Q (CoQ), we discovered that irradiation of CoQ with ultraviolet light results in the formation of a number of compounds that influence the synthesis of mevalonate pathway lipids by HepG2 cells. Among the compounds that potently stimulated CoQ synthesis while inhibiting cholesterol synthesis, derivatives of CoQ containing 1-4 epoxide moieties in their polyisoprenoid side chains were identified. Subsequently, chemical epoxidation of all-trans-polyprenols of different lengths revealed that the shorter farnesol and geranylgeraniol derivatives were without effect, whereas the longer derivatives of solanesol enhanced CoQ and markedly reduced cholesterol biosynthesis. In contrast, none of the modified trans-trans-poly-cis-polyprenols exerted noticeable effects. Tocotrienol epoxides were especially potent in our system; those with one epoxide moiety in the side-chain generally up-regulated CoQ biosynthesis by 200-300%, whereas those with two such moieties also decreased cholesterol synthesis by 50-90%. Prolonged treatment of HepG2 cells with tocotrienol epoxides for 26 days elevated their content of CoQ by 30%. In addition, the levels of mRNA encoding enzymes involved in CoQ biosynthesis were also elevated by the tocotrienol epoxides. The site of inhibition of cholesterol synthesis was shown to be oxidosqualene cyclase. In conclusion, epoxide derivatives of certain all-trans-polyisoprenoids cause pronounced stimulation of CoQ synthesis and, in some cases, simultaneous reduction of cholesterol biosynthesis by HepG2 cells.     

Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells

Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [³H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [¹⁴C]p-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. In vitro assays to measure trans-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. In vitro reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.     

Split-test Bonferroni correction for QEEG statistical maps

With statistical testing, corrections for multiple comparisons, such as Bonferroni adjustments, have given rise to controversies in the scientific community, because of their negative impact on statistical power. This impact is especially problematic for high-multidimensional data, such as multi-electrode brain recordings. With brain imaging data, a reliable method is needed to assess statistical significance of the data without losing statistical power. Conjunction analysis allows the combination of significance and consistency of an effect. Through a balanced combination of information from retest experiments (multiple trials split testing), we present an intuitively appealing, novel approach for brain imaging conjunction. The method is then tested and validated on synthetic data followed by a real-world test on QEEG data from patients with Alzheimer’s disease. This latter application requires both reliable type-I error and type-II error rates, because of the poor signal-to-noise ratio inherent in EEG signals.     

Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses.

A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)-associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low-molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7-tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC50 is approximately 20 micro m with a DNA substrate, but only 60 micro m with an RNA substrate. Several related analogues of TBBT were enzyme- and/or substrate-specific inhibitors. For example, 5,6-dichloro-1-(beta-d-ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC50 approximately 0.3 micro m), but much weaker with a DNA substrate (IC50 approximately 3 micro m). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 micro m. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,-tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP-binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP-binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.     

Evaluation of the water quality of future tributaries to the planned Dobczyce reservoir (Poland) using macroinvertebrates

The bottom fauna of 5 tributaries of the planned Dobczyce reservoir and the River Raba below the dam was investigated in the preimpoundment period (1983–84). The abundance of macrofauna varied between 46 taxa (Wolnica stream) and 66 taxa (Brzezówka stream). Each station showed individual taxa composition except for the RABA-u and RABA-d (84% similarity). On the basis of 7 different biological indices the stations were divided into 3 categories: unpolluted (Brzezówka), slightly polluted (Bulinka, Trzemésnia and both Raba stations), and moderately polluted (Wolnica). Most sensitive to chemical pollution was the BIOTIC-index. The combination of environmental variables was used to predict biological indices. The most significant relationship (P < 0.01, R ² = 0.71) was found between the BIOTIC-index and physico-chemical factors. Some problems in the application of indices (sampling, indicator organisms and interpretation of the results) are discussed and local adaptations of methods used are recommended.     

Different haplotypes for cystic fibrosis-linked DNA polymorphisms in Polish and Dutch populations

Summary We analyzed DNA from 34 Polish and 63 Dutch cystic fibrosis (CF) patients and their families using the polymorphic markers XV2c and KM19, which are in linkage disequilibrium with the CF mutation. Strong linkage disequilibrium was found in the Dutch population sample, but the haplotypes of the Polish chromosomes showed a significantly less extreme disequilibrium. Our data and previous studies indicate that the highest degree of homogeneity of the CF defect and hence the best possible use of the XV2c/KM19/CF linkage disequilibrium for CF carrier detection/exclusion is in populations of northern European origin.