Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells

Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12–18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.     

Biosorption of uranium by Myxococcus xanthus

This paper deals with uranium biosorption by Myxococcus xanthus biomass in which dry biomass, accumulating up to 2.4 mM of uranium g⁻¹, is demonstrated to be a more efficient biosorbent than wet biomass. For uranium concentrations of 0.1–0.3 mM, between 95.79% and 95.99% of the uranium was taken up from the solution. Dry biomass biosorption was found to be relatively rapid, reaching equilibrium after 5–10 min. In addition, the pH influenced biosorption, pH 4.5 promoting maximum uptake. It was also established that the biosorbed uranium is located on the cellular wall and within the extracellular mucopolysaccharide of this microorganism. Furthermore, using sodium carbonate as a desorbent agent, 80.82% of the biosorbed uranium could be recovered. The results obtained indicate the possible utilization of M. xanthus biomass to solve some problems of the water contaminated by uranium.     

The distribution of alpha-helix propensity along the polypeptide chain is not conserved in proteins from the same family.

We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of alpha-helices. We have analyzed by CD the conformation of peptides corresponding to the five alpha-helices of two alpha/beta parallel proteins (ComA and Ara). The large alpha-helical population of peptide ComA-4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21-ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275-296). Comparison of the helical content of equivalent peptides reveals that protein alpha-helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.     

The modifying effect of gibberellic acid and kinetin on the growth abnormalities induced by 2,4-D in marrow seedlings and the possible involvement of endogenous ethylene

Swelling of the hypocotyl base induced by 2,4-D in seedlings of marrow was much reduced if GA₃ was also present in the incubation medium. At appropriate concentrations kinetin also counteracted this 2,4-D effect, although at higher concentrations appeared to be ineffective. GA₃ was also able to overcome the inhibitory effects of 2,4-D on extension growth in the hypocotyl but kinetin was much less effective in this case. None of the treatments employed was able to alleviate the inhibition of radicle extension induced by 2,4-D.Ethephon induced similar responses in the seedlings to those resulting from 2,4-D treatment, while treatment with a mixture of 2,4-D and CoCl₂ removed many of these growth abnormalities. These observations are interpreted as indicating that 2,4-D operates at least partly by stimulating the production of ethylene in the tissues. 2,4-D strongly inhibited the accumulation of endogenous gibberellin during the period of seedling development examined, but enhanced cytokinin levels during the later stages of the same period. The possibility of interactions at the biosynthetic level between gibberellins, cytokinins and ethylene and their involvement in the regulation of seedling development are discussed.     

Batch culture of Candida utilis in a medium deprived of a phosphorous source

Summary Candida utilis var. major NRRL-Y-1084 was grown in a defined medium without a phosphorous (P) source. During the exponential phase, cells divided according to a specific growth rate of 0.32 h^-1, which is lower than the usual rate for a balanced medium (0.4–0.6 h^-1). The relative P content of the biomass decreased from 2.70% to 0.75% over a period of 6 h, including 2 h of cell division arrest. At the end of this period there was another interruption of cell division. After that, multiplication restarted at a considerably lower rate and it deviated slightly from the exponential pattern. The stationary phase began when biomass P content reached 0.4%–0.5%, slowly decreasing afterwards to 0.25–0.20%. Biomass synthesis was less affected than cell division by the relative decrease of endogenous P, the two processes differing partially in their kinetics. Cell lysis started shortly before the stationary phase and affected about 20% of the population by the end of the assay. RNA and P content of the resulting biomass were 2.4% and 0.25% respecitvely, P being mainly incorporated to RNA.The relationship of biomass production to glucose uptake was very low, probably because the marked P deficiency called for an increase in energy consumption for growth and specially for maintenance. Compared with yeasts grown in a balanced medium, 40% increase in glycogen was observed, whereas no mean changes in the content of cell wall carbohydrates (glucan and mannan) and that of true protein were found.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET). Agrentina     

Hydrophobic interaction chromatography of Micrococcus lysodeikticus F1-ATPase. A method to detect conformational flexibility of the molecule

Hydrophobic interaction chromatography of purified ATPase from Micrococcus lysodeikticus (E.C. 3.6.1.3.), a complex oligomeric protein, induces extensive conformational changes in it. In this report, we describe some physicochemical properties of the enzyme forms obtained. They can be summarized as follows. (1) The subunit stoichiometry of the enzyme is altered by the absorption and desorption process since most of the forms obtained are defective in gamma and delta subunits. An important reduction in the molar proportion of alpha subunit is also observed; (2) the fluorescence spectra of the different forms show progressive tyrosine residues which roughly correspond to the extent and strength of the interaction existing before elution of the enzyme; (3) circular dichroism measurements reveal changes of the secondary structure of the F1-ATPase undergoing an increase in alpha-helical content; (4) the ordered, active forms eluted from the hydrophobic chromatography columns are less stable than the native protein, as shown by dialysis experiments. These results while supporting the use of hydrophobic chromatography as a simplified model of membrane-membrane protein interaction, also indicate the need for caution in its application to the purification of complex membrane proteins.     

Glycoproteins in bacterial membranes. In vivo labeling of the sugar portion of energy-transducing ATPase and a low-molecular-weight fraction fromMicrococcus lysodeikticus membranes

The energy-transducing ATPase and a low-molecular-weight fraction ofMicrococcus lysodeikticus membranes incorporated¹⁴C label fromd-[U-¹⁴C]glucose fed to the bacteria in synthetic medium. The specific radioactivity of the sugar portion of the ATPase and low-molecular-weight fraction was, respectively, 2.65 and 2.88 times that of their amino acids. Glucose and mannose in approximately equimolar amounts were identified as the main sugars of the glycoprotein ATPase, thus confirming previous structural studies. Glucose, galactose, and mannose (1:1:2) were identified as the main sugars of the low-molecular-weight glycopeptides. These results confirm and extend the notion that glycoprotein are constituents of prokaryotic membranes.     

Biosynthetic approach to the structure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus membranes: in vivo labeling of the polypeptides and study of their relationship

The F₁-ATPase or BF₁ factor was purified from Micrococcus lysodeikticus substrain B grown in a synthetic medium in the presence of tritiated amino acids. When analyzed in sodium dodecyl sulfate-7% polyacrylamide gels, the fresh purified preparation contained α, β, γ subunits (referred as the intrinsic subunits) and two other polypeptides (designated as X and component of relative mobility 1.0) whose status as subunits remains to be established. This overall polypeptide composition was similar to that of the F₁-ATPase isolated from the same strain grown in complex medium (J. Carreira, J. M. Andreu, M. Nieto, and E. Muñoz., 1976 Mol. Cell. Biochem.10, 67–76). The distribution of ³H-labeled amino acids into purified F₁-ATPase and its constituent polypeptides under different stages of growth was used to investigate the biosynthetic relationship between the different polypeptides. The incorporation of amino acids into purified BF₁ factor was slower than that of cytoplasmic and other membrane proteins. In isotope-dilution and chase experiments, F₁-ATPase showed one of the slowest rates of decay of the incorporated label. These results point out that F₁-ATPase of M. lysodeikticus undergoes slower turnover than the overall cytoplasmic and membrane proteins. Pulse and chase experiments allowed us to conclude that the α, β, γ subunits and the components of relative mobility 1.0 are independent with differences in their turnover and therefore do not bear any apparent relation as precursors-products. The two major subunits represent seemingly the “core” of ATPase, the β subunit behaving like the most stable component. On the other hand, the γ subunit appears to be synthesized independently from this α + β complex.