Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.     

Differential patterns of serum concentration and adipose tissue expression of chemerin in obesity: Adipose depot specificity and gender dimorphism

Chemerin, a recognized chemoattractant, is expressed in adipose tissue and plays a role in adipocytes differentiation and metabolism. Gender- and adipose tissue-specific differences in human chemerin expression have not been well characterized. Therefore, these differences were assessed in the present study. The body mass index (BMI) and the circulating levels of chemerin and other inflammatory, adiposity and insulin resistance markers were assessed in female and male adults of varying degree of obesity. Chemerin mRNA expression was also measured in paired subcutaneous and visceral adipose tissue samples obtained from a subset of the study subjects. Serum chemerin concentrations correlated positively with BMI and serum leptin levels and negatively with high density lipoprotein (HDL)-cholesterol levels. No correlation was found between serum chemerin concentrations and fasting glucose, total cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides, insulin, C-reactive protein or adiponectin. Similarly, no relation was observed with the homeostasis model assessment for insulin resistance (HOMA-IR) values. Gender- and adipose tissue-specific differences were observed in chemerin mRNA expression levels, with expression significantly higher in women than men and in subcutaneous than visceral adipose tissue. Interestingly, we found a significant negative correlation between circulating chemerin levels and chemerin mRNA expression in subcutaneous fat. Among the subjects studied, circulating chemerin levels were associated with obesity markers but not with markers of insulin resistance. At the tissue level, fat depot-specific differential regulation of chemerin mRNA expression might contribute to the distinctive roles of subcutaneous vs. visceral adipose tissue in human obesity.     

Molecular diversity, metabolic transformation, and evolution of carotenoid feather pigments in cotingas (Aves: Cotingidae)

Carotenoid pigments were extracted from 29 feather patches from 25 species of cotingas (Cotingidae) representing all lineages of the family with carotenoid plumage coloration. Using high-performance liquid chromatography (HPLC), mass spectrometry, chemical analysis, and ¹H-NMR, 16 different carotenoid molecules were documented in the plumages of the cotinga family. These included common dietary xanthophylls (lutein and zeaxanthin), canary xanthophylls A and B, four well known and broadly distributed avian ketocarotenoids (canthaxanthin, astaxanthin, ??-doradexanthin, and adonixanthin), rhodoxanthin, and seven 4-methoxy-ketocarotenoids. Methoxy-ketocarotenoids were found in 12 species within seven cotinga genera, including a new, previously undescribed molecule isolated from the Andean Cock-of-the-Rock Rupicola peruviana, 3??-hydroxy-3-methoxy-??,??-carotene-4-one, which we name rupicolin. The diversity of cotinga plumage carotenoid pigments is hypothesized to be derived via four metabolic pathways from lutein, zeaxanthin, ??-cryptoxanthin, and ??-carotene. All metabolic transformations within the four pathways can be described by six or seven different enzymatic reactions. Three of these reactions are shared among three precursor pathways and are responsible for eight different metabolically derived carotenoid molecules. The function of cotinga plumage carotenoid diversity was analyzed with reflectance spectrophotometry of plumage patches and a tetrahedral model of avian color visual perception. The evolutionary history of the origin of this diversity is analyzed phylogenetically. The color space analyses document that the evolutionarily derived metabolic modifications of dietary xanthophylls have resulted in the creation of distinctive orange-red and purple visual colors.     

Correlations of trace element levels within and between different normal autopsy tissues analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES)

Imbalance in trace metal metabolism may lead to metal interactions that may be of patho-physiological importance. Knowledge of the relation between trace metals in normal tissues is needed to assess abnormal deviations associated with disease. In this study correlations between Cu, Co, Cr, Fe, Mn, Ni, Se, Zn, Al, Ba, Cd, Pb and Sr within the same and between 6 different, normal autopsy tissues were determined using Spearman rank correlation analysis based on analytical data obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES). Fe-Co were correlated in most tissues. Cu-Mn, Zn-Cu, Zn-Mn and Zn-Cd were highly correlated in the kidney medulla. Ni-Ni, Sr-Sr and Cd-Cd were correlated between several tissues, while Fe-Fe, Zn-Zn and Cu-Cu were correlated between kidney cortex and medulla. Mn-Mn was highly correlated between the liver and brain front lobe, cerebellum and heart. High correlations were found for Ni-Co and for Se-Mn between the kidney cortex and brain front lobe and pancreas respectively. Inverse correlations were found for Se-Cd between kidney cortex and cerebellum, for Se-Cd and Cd-Zn between kidney medulla and heart, for Co-Sr and Fe-Sr between the liver and kidney cortex and heart respectively, and for Sr-Mn between kidney medulla and pancreas. A large number of trace elements are statistically correlated within and between different, normal tissues. Knowledge of these correlations may contribute to increase the understanding of kinetic interactions of trace metals in the body and the role of such interactions in normal and disturbed trace metal metabolism.     

Anti-HBs antibodies from rabbits and their use as reagents for serological tests in the diagnosis of hepatitis B

By the method of affinity chromatography a partially purified antigen was obtained after passing the plasma of an asymptomatic carrier of HBsAg through a column of Sepharose 4B linked to angi-HBs. This antigen was inoculated in rabbits using a schedule of 1,0 mg in the first dose and 4 other doses of 0,5 mg with intervals of approximately 15 days. Observing that blood samples collected after the 5th inoculation showed no change in antibody levels, the animals were bled on the 62th day and these immune sera were standardized with the following tests for the detection of HBsAg: Reverse passive hemagglutination (R-PHA) - using specific gamma globulin that was obtained from rabbit sera by affinity chromatography and reaching an optimal concentration of 10 micrograms/ml to sensitise SRBC at 5% fixed in glutaraldehyde. Counter immuno electrophoresis (CIEP) - using the rabbit immune sera diluted to 1/20 as a reagent for the detection of HBsAg. The immune sera was also used to conjugate new Sepharose 4B for affinity chromatography and was found having a linking capacity of approximately 0,5 to 1,0 mg of HBsAg per ml of Sepharose after complete saturation.     

Right-handed 14-helix in beta 3-peptides from L-aspartic acid monomers

beta-Peptides made from L-aspartic acid monomers form a new class of beta 3-peptides. Here we report the first three-dimensional NMR solution structure of a beta 3-hexapeptide (1) from L-aspartic acid monomers in 2,2,2-trifluoroethanol (TFE). We show that 1 forms a right-handed 14-helical structure in TFE. alpha-peptides from naturally occurring L-amino acids adopt a right-handed alpha-helix whereas beta 3-peptides formed from beta 3-amino acids derived from naturally occurring L-amino acids form left-handed 14-helices. The right-handed 14-helical conformation of 1 is a better mimic of alpha-peptide conformations. Using the NMR structure of 1 in TFE, we further study the conformation of 1 in water, as well as two similar beta 3-peptides (2 and 3) in water and TFE by molecular dynamics (MD) simulations. NMR and MD results suggest loss of secondary structure of 1 in water and show that it forms a fully extended structure. 2 and 3 contain residues with oppositely charged side chains that engage in salt-bridge interactions and dramatically stabilize the 14-helical conformation in aqueous media.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

Regulation by glucocorticoids of cell differentiation and insulin-like growth factor binding protein production in cultured fetal rat nasal chondrocytes

Glucocorticoids (GCs) modulate insulin-like growth factor action in cartilage through mechanisms that are complex and insufficiently defined, especially in the context of cranio-facial growth. Because the family of IGF-binding proteins (IGFBP-1 to -6) is important in the regulation of IGF availability and bioactivity, we examined the effect of GCs on chondrocyte differentiation in correlation with IGFBP production in cultured fetal rat chondrocytes isolated from nasal septum cartilage of fetal rat. Dexamethasone (DEX) effects were tested before and at the onset of extracellular matrix maturation. DEX induced a dose-dependent increase in the size of cartilage nodule formed, (45)Ca incorporation into extracellular matrix, alkaline phosphatase activity, and sulfatation of glycosaminoglycans, maximal effects being obtained with a 10-mM DEX concentration. The IGFBPs produced by cultured chondrocytes were characterized in culture medium which had been conditioned for 24 h under serum-free conditions by these cells. Western ligand blotting with a mixture of [(125)I]IGF-I and -II revealed bands of 20, 24, 29, a 31-32 kDa doublet and a 39-41 kDa triplet which were differently regulated by DEX. Immunoblotting showed that following DEX exposure, IGFBP-3 and -6 were up-regulated whereas IGFBP-2, -5, and the 24 kDa band were down-regulated. The effect of DEX on both differentiation and IGFBP production showed a same dependence, and developed when extracellular matrix maturation had been just induced. The results obtained in this chondrocyte culture system show that production of IGFBPs is modulated by DEX at physiological concentrations thus regulating IGF availability and action, a control which could promote the primordial role of the rat nasal septum in craniofacial growth.     

Preparation of transparent photoluminescence smart window by integration of rare-earth aluminate nanoparticles into recycled polyethylene waste

Novel photoluminescent nanocomposite sheets were prepared for simple commercial manufacturing of transparent and luminous photochromic smart windows. A simple physical integration of lanthanide-doped strontium aluminium oxide (LdSAO) nanoparticles into recycled polyethylene (PE) waste produced a smart nanocomposite with persistent phosphorescence and photochromic properties. Because the nanoparticle form of LdSAO is important for developing transparent materials, LdSAO nanoparticles were well dispersed in the PE matrix. Both the morphologies and chemical compositions of the LdSAO nanoparticles and LdSAO-containing luminescent PE sheets were investigated. Both LdSAO-free and photoluminescent PE sheets were colourless in normal daylight. However the LdSAO-containing PE luminescent samples only exhibited a brilliant green colour under ultraviolet (UV) light and a greenish-yellow colour in the dark as verified by Commission Internationale de l'éclairage laboratory parameters. Both absorbance and emission bands were monitored at 377 and 436/517 nm, respectively. The LdSAO-containing PE luminescent sheets were compared with the LdSAO-free sample using both photoluminescence spectroscopy and for their mechanical properties and were found to have improved scratch resistance, UV protection, and superhydrophobic activity. Due to the added LdSAO, photoluminescence, decay, and lifetime spectral tests confirmed its photochromic fluorescence and long-lasting phosphorescence characteristics. The PE@LdSAO nanocomposite sheets displayed UV protection, photostability, hydrophobicity, and excellent durability compared with the blank LdSAO-free PE sheet.     

Degradation of salicylic acid by Fusarium graminearum

Fusarium head blight (FHB) is a major cereal crop disease, caused most frequently by the fungus Fusarium graminearum. We have previously demonstrated that F. graminearum can utilize SA as sole source of carbon to grow. In this current study, we further characterized selected four fungal SA-responsive genes that are predicted to encode salicylic acid (SA)-degrading enzymes and we used a gene replacement approach to characterize them further. These included two genes predicted to encode a salicylate 1-monooxygenase, FGSG_03657 and FGSG_09063, a catechol 1, 2-dioxygenase gene, FGSG_03667, and a 2, 3-dihydroxybenzoic acid decarboxylase gene, FGSG_09061. For each gene, three independent gene replacement strains were assayed for their ability to degrade salicylic acid in liquid culture. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were shown to be essential for SA degradation, while a loss of 2, 3-dihydroxybenzoic acid decarboxylase FGSG_09061 caused only a partial reduction of SA degradation and a loss of salicylate 1-monooxygenase FGSG_09063 had no effect when compared to wild type culture. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were identified as the first two key enzyme steps of SA degradation via catechol in the β-ketoadipate pathway. Expression profiles for all four genes were also determined in liquid culture and in planta. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were co-expressed and their expression was substrate dependent in liquid culture; however their expression was uncoupled in planta. Disruption of the gene for catechol 1, 2-dioxygenase FGSG_03667 was shown to have no effect on fungal virulence on wheat. Our results with 2, 3-dihydroxybenzoic acid decarboxylase FGSG_09061 raise the possibility of an alternate non-oxidative decarboxylation pathway for the conversion of SA to catechol via 2, 3-dihydrozybenzoic acid and for a connection between the oxidative and the non-oxidative decarboxylation pathways for SA conversion.