THE UNKNOWN SHADOWS OF TREHALASE.

Each year brings new facts concerning multiple roles of sugar pathways in plant metabolism. One of them--the trehalose pathway--has been shown to play a role in stress signalling. The last enzyme of this pathway--trehalase--has been proven to be strongly expressed in guard cells. Modifications of its abundance cause changes in stomatal closure and response to abscisic acid. Our phenotypical studies of different mutants of Arabidopsis thaliana and Musa have enabled us to propose a new function of trehalase. It might play a role in the feedback of sucrose as a closing signal for stomata in reaction to an efficient photosynthesis. To characterize the phenotype we measured: the dynamic cumulative water loss, the dynamic leaf surface temperature, and the stomatal conductance. Based on the obtained results we have determined the time points for a proteomics study. The exact role of trehalase and related proteins in the proposed mechanism will be defined with multiple analysis including mass spectrophotometry and enzymatic activities. The samples will be collected from a wide type of plants including model organism (Arabidopsis--wild type, trehalase mutant plants) and crops (banana). The final results will shed light on the complete role of trehalase and the feedback pathway.

p-Coumaric acid loaded into liquid crystalline systems as a novel strategy to the treatment of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is an extremely common type of vaginal infection, which is mainly caused by Candida albicans. However, non-albicans Candida species are frequently more resistant to conventional antifungal agents and can represent up to 30% of cases. Due to side effects and increasing antifungal resistance presented by standard therapies, phenolic compounds, such as p-coumaric acid (p-CA), have been studied as molecules from natural sources with potential antifungal activity. p-CA is a poorly water-soluble compound, thus loading it into liquid crystals (LCs) may increase its solubility and effectiveness on the vaginal mucosa. Thereby, here we propose the development of mucoadhesive liquid crystalline systems with controlled release of p-CA, for the local treatment of VVC. Developed LCs consisted of fixed oily and aqueous phases (oleic acid and cholesterol (5:1) and poloxamer dispersion 16%, respectively), changing only the surfactant phase components (triethanolamine oleate (TEA-Oleate) or triethanolamine (TEA), the latter producing TEA-Oleate molecules when mixed with oleic acid). Systems were also diluted in artificial vaginal mucus (1:1 ratio) to mimic the vaginal environment and verify possible structural changes on formulations upon exposure to the mucosa. From the characterization assays, p-CA loaded TEA-Oleate systems presented mucoadhesive profile, liquid crystalline mesophases, well-organized structures and pseudoplastic behaviour, which are desirable parameters for topical formulations. Moreover, they were able to control the release of p-CA throughout the 12 h assay, as well as decrease its permeation into the vaginal mucosa. p-CA showed antifungal activity in vitro against reference strains of C. albicans (SC5314), C. glabrata (ATCC 2001) and C. krusei (ATCC 6258), and exhibited higher eradication of mature biofilms than amphotericin B and fluconazole. In vivo experiments demonstrated that the formulations reduced the presence of filamentous forms in the vaginal lavages and provided an improvement in swelling and redness present in the mice vaginal regions. Altogether, here we demonstrated the potential and feasibility of using p-CA loaded liquid crystalline systems as a mucoadhesive drug delivery system for topical treatment of VVC.

Parameters affecting ethyl ester production by Saccharomyces cerevisiae during fermentation.

Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.

The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling.

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1(ala252) allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Delta. The difference between the Pde1(ala252) allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2(val19) pde2Delta background, the Pde1(ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1Delta, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1(ala252) mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1(ala252), and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana.

Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role. Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants. We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases. A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast. Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose.

Negatively charged phospholipids and their position in the cholesterol affinity sequence.

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyeline with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin. From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin greater than phosphatidylserine, phosphatidylglycerol greater than phosphatidylcholine greater than phosphatidylethanolamine.

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.

Influence of Ca2+ and Mg2+ on the thermotropic behaviour and permeability properties of liposomes prepared from dimyristoyl phosphatidylglycerol and mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine.

Calorimetric experiments showed a marked effect of Ca2+ and Mg2+ on the thermotropic behaviour of dimyristoyl phosphatidylglycerol. 2. Concentrations of Ca2+ and Mg2+ lower than 1 ion to 2 molecules of phosphatidylglycerol produced a shift of the phase transition to higher temperatures and an increase in the enthalpy change which is consistent with a closer packing of the lipid molecules in the liposomes. 3. Above the 1:2 ratio, freeze-fracture electron microscopy demonstrated typical "crystal" structures both in the presence of Ca2+ and Mg2+. In the presence of Mg2+ a metastable behaviour was noticed in the calorimetric experiments. 4. A Ca2+- and Mg2+-induced shift in the transition temperature and an increase in the enthalpy change was also observed in a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine. However, these mixed samples remained liposomal in structure at any concentration of the divalent ions. 5. Liposomes prepared from a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the absence of divalent cations are permeable in the range 10-50 degrees C. Bilayers of mixtures neutralized by Ca2+ or Mg2+ were demonstrated to be completely impermeable to K+, except in the vicinity of the phase transition. 6. The leak of ions from liposomes of a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the vicinity of the phase transition temperature was considerably less in the presence of Ca2+ than in the presence of Mg2+. 7. It is concluded that there is a correlation between the calorimetric data and the permeability properties of dimyristoyl phosphatidylglycerol-containing bilayers with respect to the influence of Ca2+ and Mg2+.

Phase transitions in phospholipid model membranes of different curvature.

1. Nuclear magnetic resonance, light scattering and freeze fracturing electron microscopic techniques were used to characterize the size of unilamellar phospholipid vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. 2. Differential scanning calorimetric and light scattering analyses showed that very small unilamellar vesicles obtained by the sonication method exhibit a downward shifted, largely broadened phase transition with a slightly decreased enthalpy change when compared with multilayered liposomes. 3. The phase transition of vesicles with variable diameter as obtained by injection methods resembled the pattern of multilayered liposomes the more the diameter was increased. 4. Repeated cycling through the lipid phase transition was shown to have a progressive effect on a fusion process. This effect was strongly increased when the osmolarity of the medium was enhanced (e.g. by the addition of cryoprotectors). Furthermore it was shown that ice-water of the systems caused abrupt fusion of the lipid structures. 5. Controversial results in the literature on the thermotropic behavior of vesicles could be explained in terms of these fusion processes.

Miscibility properties of binary phosphatidylcholine mixtures. A calorimetric study.

From data obtained by differential scanning calorimetry phase diagrams were constructed, using a thermodynamically based fitting method. The following binary mixtures of phosphatidylcholines in water were studied: 14:0/14:0-glycerophosphocholine/16:0/16:0-glucerophosphocholine, 14:0/14:0-glycerophosphocholine/18:0/18:0-glycerophosphocholine, 12:0/12:0-glycerophosphocholine/16:0/16:0-glycerophosphocholine, 18:1t/18:1t-glycerophosphocholine/14:0/14:0-glycerophosphocholine and 18:1t/18:1t-glycerophosphocholine/16:0/16:0-glycerophosphocholine. A comparison is made of the present results with those obtained using probe techniques and the differences are discussed.

The preferential interaction of cholesterol with different classes of phospholipids.

1. By differential scanning calorimetry a preferential affinity of cholesterol for sphingomyelin was established in mixtures of sphingomelin and phosphatidylcholine where sphingomyelin was either the higher or the lower melting phospholipid. 2. A preferential affinity of cholesterol for sphingomyelin was also found in mixtures of sphingomyelin and phosphatidylethanolamine where sphingomyelin was either the higher or the lower melting phospholipid. The sphingomyelin used was isolated from beef erythrocytes or synthetic palmitoyl sphingomyelin. 3. In mixtures of phosphatidylserine with phosphatidylethanolamine, or phosphatidylserine with phosphatidylcholine, cholesterol showed the highest affinity for the lower melting phospholipid. 4. In a previous paper (van Dijck et al. (1976) Biochim. Biophys. Acta 455, 576-588) it was established that cholesterol has a higher affinity for phosphatidylcholine than for phosphatidylethanolamine. The affinity order of cholesterol for the neutral phospholipids which can be deduced form these experiments is sphingomyelin greater than phosphatidylcholine greater than phosphatidylethanolamine.

The interaction of spectrin - actin and synthetic phospholipids.

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin - actin complex isolated from human erythrocyte membranes. With dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca2+ and Mg2+ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.

The preference of cholesterol for phosphatidylcholine in mixed phosphatidylcholine-phosphatidylethanolamine bilayers.

The following phosphatidylethanolamines were studied by differential scanning calorimetry: 1,2-dipalmitoleoyl-, 1,2-dioleoyl-, 1,2-dilauroyl-, 1,2-dielaidyl-, 1,2-dimyristoyl- and 1,2-dipalmitoyl-sn-glycero-3-phosphoryl-ethanolamine. The saturated and trans-unsaturated species underwent thermotropic phase transitions at temperatures about 20-30 degrees C higher than the corresponding phosphatidylcholines but the enthalpy changes were nearly identical. The transition temperatures for the cis-unsaturated species were about the same as those of the corresponding phosphatidylcholines but here the enthalpy change was markedly decreased as compared with the phosphatidylcholines. Freeze-fracture electron microscopy revealed phase changes from a lamellar to a hexagonal phase for 1,2-dipalmitoleoyl- and 1,2-dioleoyl-sn-glycero-phosphorylethanolamine at 20 and 0 degrees C respectively. At these temperatures no transitions were apparent in the calorimeter scan. Incorporation of increasing amounts of cholesterol into phosphatidylethanol-amine bilayers gradually decreased the enthalpy changes of the phase transition in the same manner as was demonstrated before for phosphatidylcholine/cholesterol mixtures. This was studied both for 1,2-dipalmitoleoyl- and 1,2-dimyristoyl-sn-glycerophosphorylethanolamine. In an equimolar mixture of 1,2-dioleoyl- and 1,2-dipalmitoylphosphoryl-ethanolamine, which showed phase separation, cholesterol preferentially decreased the transition of the lowest melting component. In equimolar mixtures of phosphatidylethanolamines and phosphatidylcholines, which showed phase separation, cholesterol preferentially abolished the transition of the phosphatidylcholine component present. This occurred both in experiments where the phosphatidylcholine was the lowest melting and where it was the highest melting component present in the mixture. These experiments strongly suggest that in phosphatidylcholine-phosphatidylethanolamine mixtures at temperatures where both components are in the liquid-crystalline state cholesterol is preferently associated with the phosphatidylcholine component in the mixture.

Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine.

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.

Effect of glycophorin incorporation on the physico-chemical properties of phospholipid bilayers.

1. The thermotropic behaviour of phospholipid molecules in reconstituted glycophorin-containing vesicles has been investigated by means of differential scanning calorimetry. Each glycophorin molecule is able to perturb the properties of 80--100 phospholipid molecules in such a way that these lipid molecules no longer participate in the cooperative gel to liquid-crystalline phase transition. This number of perturbed phospholipid molecules was discovered to be independent of the lipid charge. 2. By means of freeze-facture electron microscopy it could be demonstrated that glycophorin is not excluded from the solid lipid phase upon cooling the lipids below their gel to liquid-crystalline phase transition temperature. In mixtures of phosphatidylcholines which show solid-solid immiscibility, glycophorin is preferentially associated with the lower-melting lipid component upon phase separation, as could be demonstrated by both differential scanning calorimetry and freeze-fracture electron microscopy. 3. The effect of glycophorin on the mobility of phospholipids has been investigated by means of 31 P NMR. Glycophorin, incorporated into sonicated vesicles of dioleoylphosphatidic acid, is able to immobilize nine lipid molecules very strongly in their phosphate region. Evidence for an electrostatic inter-action between the protein and this negatively charged phospholipid has been presented. 4. The presence of glycophorin causes discontinuities in the lipid bilayer. This results in higher susceptibility of the bilayer towards attack by lipolytic enzymes and in enhanced membrane permeability.

Fungal pathogens research: novel and improved molecular approaches for the discovery of antifungal drug targets.

With the rise of fungal infection incidence amongst the patient population, the importance of developing new antifungal drug targets is higher than ever. This review mainly focuses on the three most prevalent fungal pathogens, Candida, Aspergillus and Cryptococcus, and on the most recent progresses in molecular research that contribute to a better understanding of the pathogen itself, but also its host and the interaction with its host. We consider the progress made in comparative genomics following the huge effort of fungal genome sequence projects undertaken in the last few years. We focus not only on currently used mammalian animal models such as mice, but also on novel non-mammalian models, such as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, which offer useful tools in the area of the innate immune response to fungal infections. In addition we relate to the recent genomic and proteomic studies and focus on the use of these approaches in in vivo experiments in the pathogen itself as well as in the host. Finally, we describe the latest targeted mutagenesis strategy available in C. albicans and the use of RNA interference in both Cryptococcus neoformans and A. fumigatus. Our aim is not to give an exhaustive list of all new strategies but rather to give an overview of what will contribute most to the identification of new antifungal drug targets and the establishment of novel antifungal strategies.

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin.

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.

Kallikrein-related protease in the rat ventral prostate: cDNA cloning and androgen regulation.

A kallikrein-related protease was purified from rat ventral prostate cytosol by means of DEAE-Sepharose chromatography, followed by gel filtration on Sephadex G-100 and CM-cellulose chromatography. Antibodies raised in rabbits against the purified protease recognize two bands on immunoblots of prostatic cytosol: a 31,000 Da band and an 18,000 Da band, which constitutes a proteolytic breakdown product of the former. The corresponding cDNA was isolated from a prostatic cDNA library, inserted in a lambda gt11 vector, using immunodetection for screening and identified as encoding a kallikrein- and tonin-related protease. Castration resulted in a marked decrease of the level of the protease and its mRNA, whereas administration of androgens to castrated animals resulted in marked stimulation. These data support the hypothesis that this protease is a member of a cluster of proteins, that are regulated in parallel by androgens in prostatic epithelial cells.

Binding of androgen-receptor complexes to alpha 2u-globulin genes and to the long terminal repeat of mouse mammary tumor virus.

The binding of androgen-receptor complexes to fragments derived from two alpha 2u-globulin genes (RAP 01 and RAO 01) was studied using a DNA-cellulose competition assay. Rat prostate cytosol labelled with [3H]mibolerone was used as a source of the androgen receptor. Two controls were included in these studies: the long terminal repeat (LTR) of mouse mammary tumor virus which has previously been shown to act as an androgen response element and a fragment of the C3 gene of prostatic binding protein which has been demonstrated to bind androgen-receptor complexes. Our experiments indicate that androgen-receptor complexes bind specifically and with comparable affinity to the C3 gene fragment, the LTR and a fragment of RAP 01 located in the 5'-upstream region (bp -642 up to -584). No specific interaction was observed with fragments derived from RAO 01. The region of RAP 01 which binds androgen-receptor complexes has previously been shown to interact with glucocorticoid receptors and contains a 17 bp sequence homologous with the consensus sequence for glucocorticoid-receptor binding. A mutation in this sequence in RAO 01 may be responsible for the loss of glucocorticoid and androgen-receptor binding. It is concluded that at least one member of the alpha 2u-globulin gene family interacts directly with androgen-receptor complexes with an affinity comparable to that observed for other androgen-dependent genes. The binding is observed in a region displaying also affinity for the glucocorticoid receptor.

Interaction of the 90-kDa heat shock protein with native and in vitro translated androgen receptor and receptor fragments.

Androgen receptor (AR) from rat ventral prostate and AR synthesized in vitro by translation in rabbit reticulocyte lysate of AR mRNA, transcribed from a pGEM-4Z DNA template were compared by gel permeation chromatography and by sucrose gradient ultracentrifugation. Under non-activating conditions the AR from rat prostate migrated as an 8-9 S complex of approx. 300 kDa. The addition of chicken antibodies against HSP90 shifted this complex to the void volume of the column or to the bottom of the ultracentrifugation gradient. Under activating conditions, on the other hand, the AR migrated as a 110 kDa, 5.2 S protein and was no longer displaced by HSP90 antibodies. Under all these conditions, the behaviour of in vitro synthesized AR was very similar to that of AR from rat prostate. By selective use of restriction enzymes on the template of transcription AR mutants could be prepared from which an increasing part was deleted at their carboxy terminal end. The interaction with HSP90 was conserved for AR1-758 missing the last 145 amino acids, but was lost in AR1-703. Furthermore, a large internal deletion (ARd41-469) of the major part of the amino terminal half of the AR did not result in the loss of HSP90 binding. These results indicate that a specific subregion (amino acids 704-758) of the carboxy terminal half of the AR is required for the interaction with HSP90.