Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes

Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.     

Availability of phosphorus and sulfur of insecticide origin by fungi

Thirteen fungal species isolated from soil treated with pesticides were tested for their ability to mineralize and degrade three organophosphate insecticides currently used in Egypt (Cyolan®, Malathion® and Dursban®) in liquid media free from phosporus (P) and sulfur (S). All fungal species grew successfully on the culture media treated with the three used doses of insecticides (10, 50 and 100 ppm active ingredient) but the growth rate varied with the species, the insecticide and the doses. At 10 ppm level, insecticide degradation expressed in term of organic P mineralization (calculated as % of applied P) was the highest with all fungi tested. Organic P mineralization from pesticides was decreased by increasing the dose used to 50 and 100 ppm. The highest amount of P mineralized was observed with Cyolan® followed by Malathion® whilst P mineralization from Dursban® proceeded very slowly. Aspergillus terreus showed the greatest potential to mineralize organic P followed by A. tamarii, A. niger, Trichoderma harzianum and Penicillium brevicompactum whilst the remaining fungi only moderately mineralized the organic P component of the insecticides tested. Organic sulfur mineralization by the used fungal species paralleled, to some extent, organic P mineralization. The extracellular protein content of culture filtrates in the presence of various doses of insecticides was also decreased by increasing insecticide concentrations. The extracellular protein was significantly correlated with P and S mineralization (r = 0.89** and 0.64**, respectively) whilst correlation with cell dry mass was not significant (r = 0.03 and 0.003) suggesting a direct relationship between pesticide degradation and microbial protein production. The addition of P or S to the growth media enhanced extracellular protein excretion, and increased organic P and S mineralization by the most potent species tested (A. niger, A. tamarii, A. terreus and T. harzianum). This increment was significant in most cases, especially at the higher application rates. The relationship between extracellular protein excretion and organic P and S mineralization from insecticides was highly significant with the addition of inorganic phosphorus (r = 0.96** and 0.83**, respectively) or sulfur (r = 0.85** and 0.89**, respectively) to the growth media.     

Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1

Recent studies have demonstrated that the ATP-binding cassette transporter A1 (ABCA1) facilitates the efflux of phospholipids and cholesterol to apoprotein acceptors, leading to the synthesis of HDL. The purpose of this study was to determine the changes in the lipoprotein fractions in Abca1-deficient mice and study the mechanisms responsible for the low levels of HDL when ABCA1 is absent. Plasma phospholipid concentration was decreased by more than 75%, mostly due to a reduction of phosphatidylcholine (PC) in HDL. Abca1(-/-) HDL represents less than 2% of wild-type levels and is smaller and enriched in phospholipids (11.2-fold more than HDL from controls). Compared to wild-type littermates, Abca1(-/-) HDL had a 4-fold increase in PC, whereas lysophosphatidylcholine (LPC) (125-fold), sphingomyelin (SPH) (49-fold), and phosphatidylethanolamine (PE) (18-fold) showed even higher increases. As a consequence, the ratios of LPC/PC, SPH/PC, PE/PC, and phosphatidylinositol + phosphatidylserine (PI+PS)/PC were all much higher in HDL from Abca1(-/-), compared to wild-type HDL. Plasma phospholipid transfer protein (PLTP) and lecithin cholesterol acyltransferase (LCAT) activities were decreased by more than 80%, suggesting that the maturation of HDL is affected. To test this hypothesis, plasma from Abca1(-/-) mice was incubated with CHO cells that are known to express high levels of ABCA1 with the intent of restoring the flux of phospholipid and cholesterol onto apoAI. Compared to native plasma, no change in maturation of HDL was observed. In contrast, a 220% increase in the formation of mature HDL was observed when ABCA1 function and LCAT activities were restored. Taken together, these observations suggest that ABCA1 is necessary for the adequate lipidation of apoAI, which enables the interaction with LCAT and subsequent maturation.     

Phylogenetic position of Mediterranean Astereae and character evolution of daisies (Bellis,Asteraceae) inferred from nrDNA ITS sequences

Phylogenetic analyses of nrITS sequences of Asteraceae revealed that the Bellis group is a natural assemblage comprising all the species of Bellis and Bellium, but not Rhynchospermum. In contrast, we propose to include the genera Bellis, Bellium, and Bellidastrum in the subtribe Bellidinae in the interest of circumscribing natural groups. Our results also suggest an early diversification in the western Mediterranean Basin of two monophyletic lineages, Bellis and Bellium. Three major groups can be distinguished within BELLIS: (1) the B. perennis group, containing five annual and perennial species with three ploidy levels (diploid, octoploid, and decaploid), which are distributed throughout the Mediterranean Basin despite lack of pappus; (2) the Bellis sylvestris group, with five annual and perennial species primarily from the western Mediterranean, in which there are five ploidy levels (diploid, tetraploid, hexaploid, octoploid, and decaploid); and (3) a basal grade consisting of three diploid, perennial species which displays remarkable diversification of morphologies. Striking characteristics, such as an annual life form, polyploidy, and loss of pappus, seem to have occurred in parallel several times and in different geographical areas during the early diversification of Bellis species in the western Mediterranean. Character evolution reconstructions allow us to describe a putative ancestor of the genus Bellis (proto-Bellis).     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6)

Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues. The normal function of MRP6, including its physiological substrate(s), remains unknown. To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris. The protein was expressed in the membrane fraction as a stable 170 kDa protein. Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-[alpha-(32)P]ATP and compared to those of the drug efflux pump MRP1. rMrp6 can bind 8-azido-[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion. Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-[alpha-(32)P]ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot. Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-[alpha-(32)P]adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion. This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-[alpha-(32)P]ADP to the protein. By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins. The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, [(125)I]-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds. The P. pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6. In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in [(125)I]-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

Gastric mucosal integrity: gastric mucosal blood flow and microcirculation. An overview

The stomach is in a state of continuous exposure to potentially hazardous agents. Hydrochloric acid together with pepsin constitutes a major and serious threat to the gastric mucosa. Reflux of alkaline duodenal contents containing bile and pancreatic enzymes are additional important injurious factors of endogenous origin. Alcohol, cigarette smoking, drugs and particularly aspirin and aspirin-like drugs, and steroids are among exogenous mucosal irritants that can inflict mucosal injury. The ability of the stomach to defend itself against these noxious agents has been ascribed to a number of factors constituting the gastric mucosal defense. These include mucus and bicarbonate secreted by surface epithelial cells, prostaglandins, sulfhydryl compounds and gastric mucosal blood flow. The latter is considered by several researchers to be of paramount importance in maintaining gastric mucosal integrity. The aim of this paper is to review the experimental and clinical data dealing with the role of mucosal blood flow and in particular the microcirculation in both damage and protection of the gastric mucosa.