Adrenal glands and testes as steroidogenic tissue are affected by retinoylation reaction

This study was undertaken to better understand the physiological role of the retinoylation process in steroidogenic tissues. In adrenal gland mitochondria, the retinoylation extent was found equal to that of testes mitochondria but without ATP in the incubation buffer. We pointed out that the endogenous mitochondrial ATP in adrenal glands is much higher than in testes, about 1.3 x 10⁻² M and 5.2 x 10⁻⁸ M, respectively. In addition, less CoASH is required for the maximal acylation activity of the retinoyl moiety to protein(s) compared to testes. The fatty acid analysis revealed a different composition of mitochondrial membranes of these two tissues. Among the different values of fatty acids, it is important to note that adrenal glands contain a much higher amount of C18:0 and a much lower amount of C22:5 ω6 and C22:6 ω3 than testes in the mitochondrial membranes. In addition, there were also differences in arachidonic acid (ARA, C20:4 ω6) content between adrenal glands and testes mitochondria. These different values in the fatty acids composition should explain the different extent of the retinoylation process between the two organs.     

Overexpression of homogeneous and active ABCG2 in insect cells

The multidrug transporter ABCG2, a membrane protein with six transmembrane segments, can be overexpressed with the baculovirus/insect cell system. However, ABCG2 is produced as two species with different migration behavior via SDS–PAGE. Evidences suggest that this is due to the accumulation of an immature ABCG2 species, since: (i) the upper species, with higher apparent molecular weight, was favored by treatments reducing the rate of protein synthesis; (ii) the lower species was accumulated in presence of an endoplasmic reticulum stress inducer, and could be converted into the upper species during electrophoresis with 9 M urea; (iii) each species was differently solubilized by detergents: the upper species was partially solubilized by non-ionic and zwitterionic detergents, whereas the lower one required stronger surfactants; (iv) membrane ATPase activity from infected insect cells was essentially associated to the upper species. Altogether, these results suggest that although the insect cell/baculovirus system is not ideally adapted to overexpress human ABCG2, it is able to produce appreciable amounts of purified protein and the addition of agents reducing the rate of protein synthesis improves the homogeneity, making it a suitable heterologous expression system.     

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi^Met binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi^Met, which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.     

How to cope with the quest for new antibiotics

Since their introduction in therapy, antibiotics have played an essential role in human society, saving millions of lives, allowing safe surgery, organ transplants, cancer therapy. Antibiotics have also helped to elucidate several biological mechanisms and boosted the birth and growth of pharmaceutical companies, generating profits and royalties. The golden era of antibiotics and the scientific and economical drive of big pharma towards these molecules is long gone, but the need for effective antibiotics is increased as their pipelines dwindle and multi-resistant pathogenic strains spread. Here we outline some strategies that could help meet this emergency and list promising new targets.     

Cadmium retention in rice roots is influenced by cadmium availability, chelation and translocation

Analysis of rice plants exposed to a broad range of relatively low and environmentally realistic Cd concentrations showed that the root capacity to retain Cd ions rose from 49 to 79%, corresponding to increases in the external Cd2+ concentration in the 0.01-1 μM range. Fractioning of Cd ions retained by roots revealed that different events along the metal sequestration pathway (i.e. chelation by thiols, vacuolar compartmentalization, adsorption) contributed to Cd immobilization in the roots. However, large amounts of Cd ions (around 24% of the total amount) predictable as potentially mobile were still found in all conditions, while the amount of Cd ions loaded in the xylem seemed to have already reached saturation at 0.1 μM Cd2+, suggesting that Cd translocation may also play an indirect role in determining Cd root retention, especially at the highest external concentrations. In silico search and preliminary analyses in yeast suggest OsHMA2 as a good candidate for the control of Cd xylem loading in rice. Taken as a whole, data indicate Cd chelation, compartmentalization, adsorption and translocation processes as components of a complex 'firewall system' which acts in limiting Cd translocation from the root to the shoot and which reaches different equilibrium positions depending on Cd external concentration.     

A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.     

Triterpenoids of Terminalia sericea

The structures of sericic acid and sericoside, the major constituents of the roots of Terminalia sericea, have been determined. Sericic acid is 2α,3β,19α,24-tetrahydroxy-olean-12-en-28-oic acid and sericoside the corresponding C-28 D-glucosyl ester.