Adipose tissue R2* signal is increased in subjects with obesity: A preliminary MRI study

Objective : Circulating and adipose tissue markers of iron overload are increased in subjects with obesity. The aim is to study iron signals in adipose tissue. Methods: Adipose tissue R2* values and hepatic iron concentration (HIC) were evaluated using magnetic resonance imaging (MRI) in 23 middle‐aged subjects with obesity and 20 subjects without obesity. Results: Subcutaneous (SAT) and visceral adipose tissue (VAT) R2* were increased in subjects with obesity (P = 0.004 and P = 0.008) and correlated significantly and positively with HIC in all subjects. Strikingly, most of the associations of liver iron with metabolic parameters were replicated with SAT and VAT R2*. BMI, waist circumference, fat mass, HOMA value, and C‐reactive protein positively correlated with HIC and SAT and VAT R2*. BMI or percent fat mass (but not insulin resistance) contributed independently to 26.8‐34.8% of the variance in sex‐ and age‐adjusted SAT or VAT R2* (β > 0.40, P < 0.005). Within subjects with obesity, total cholesterol independently contributed to 14.8% of sex‐ and age‐adjusted VAT iron variance (β = 0.50, P = 0.025). Conclusions: Increased R2* in adipose tissue, which might indicate iron content, runs in parallel to liver iron stores of subjects with obesity. VAT iron seems also associated with serum cholesterol within subjects with obesity.     

Proliferation kinetics of MCF-7 cell cultures. II. Synchronization and cell recruitment induced by hormones

The culture of MCF-7 cells in well known experimental conditions and the analysis of the cellular uptake of 3HTdR under the influence of estrogens (17-beta-E2) and antiestrogens (OH-TAM) at precise concentration levels (10(-6)/10(-9) M) have made possible the study of the growing kinetic process of the cellular cultured population and the determination of the action of such hormonal preparations on the same process. The main findings are as follows: the growing rate of the cultured cells appears to slow down in presence of OH-TAM; the reason for this slowing growth seems to be the "blockade" of the cultured cells in the last part of G1 phase, a phenomenon that has proved to be dose-dependent; by the stimulating effect of the 17-beta-E2, the MCF-7 cells, synchronized in G1, progress simultaneously in their vital cycle during, at least, three divisory cycles; and this "recruitment" effect seems to be characteristic of the estrogen since the synchronization process fades or disappears when the 17-beta-E2 is absent from the culture medium.     

Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at ?20 °C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at ?20 °C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.     

A GDPase/UDPase bifunctional enzyme from Candida albicans: purification and biochemical characterization

The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.

     

Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.     

Ubiquitous urease affects soybean susceptibility to fungi

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.     

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.