Long-incubation-time gamma interferon release assays in response to purified protein derivative, ESAT-6, and/or CFP-10 for the diagnosis of Mycobacterium tuberculosis infection in children.

The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-γ) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-γ response to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.

[Proteomics studies on arthritis by SELDI-TOF-MS: identification of the S100 proteins family as proteins of interest]. / Protéomique par SELDI-TOF-MS des maladies inflammatoires articulaires: identification des protéines S100 comme protéines d'intérêt.

Clinical proteomics is a technical approach studying the entire proteome expressed by cells, tissues or organs. It describes the dynamics of cell regulation by detecting molecular events related to diseases development. Proteomic techniques focus mainly on identification of new biomarkers or new therapeutic targets. It is a multidisciplinary approach using medical, biological, bioanalytical and bioinformatics knowledges. A strong collaboration between these fields allowed SELDI-TOF-MS proteomics studies to be performed at the CHU and the University of Liège, in GIGA-Research facilities. The aim of these studies was driven along three main axes of research related to the identification of biomarkers specific to a studied pathology, to a common biological pathway and, finally, to a treatment response. This work was presented in the setting of the "Synthèse CHU 2009" meeting.

The photoreceptive capacity of the developing pineal gland and eye of the golden hamster (Mesocricetus auratus).

Anatomical and physiological studies have suggested that the pineal gland of neonatal mammals has a photoreceptive capacity. Using the golden hamster (Mesocricetus auratus) as our model, we applied biochemical approaches to look for a functional photopigment within the pineal during early development. Immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) were used to localize and quantify opsin, and high-performance liquid chromatography (HPLC) to identify photopigment chromophore (11-cis and all-trans retinaldehyde) in the developing eye and pineal. For HPLC analysis, retinaldehydes were converted to their corresponding retinoid oximes. Eluted retinoids were identified by comparison with standard vitamin A1 retinoid oxime isomers on the basis of relative elution sequence and characteristic absorbance spectra. Both immunocytochemistry and ELISA suggested an increase in the opsin content of the pineal during the first week of life. In the eye, 11-cis retinaldehyde was first detected between days 3 and 5 after birth. In three separate extractions, and using a considerable excess of pineal tissue, we failed to identify chromophore within the pineal during the first week of postnatal development. The appearance of 11-cis retinaldehyde within the eye between postnatal days 3-5 is consistent with the hypothesis that retinol isomerase activity is coordinated with outer segment development. The failure to identify chromophore within the neonatal pineal suggests that this gland lacks a functional opsin-based photopigment. These data contradict physiological evidence suggesting that the neonatal pineal of mammals contains photoreceptors.

Isolation and characterization of the activated B-cell factor 1 homolog in Caenorhabditis elegans.

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate a wide array of developmental processes in many cell types, including cell fate specification, differentiation and morphogenesis. Our studies describe the cloning of a gene from the nematode Caenorhabditis elegans that is closely related to the vertebrate-activated B-cell factor (ABF) gene. The nematode gene product CeABF-1 was detected by northern blot analysis from RNA isolated from pooled nematodes representing different developmental stages. The developmental expression profile of CeABF-1 was shown by RT-PCR analysis to be predominantly expressed in the larval stages L3 and L4, with lower levels observed in the L2 larval stage and adult. We also show that CeABF-1 is capable of forming heterodimers with E2A proteins and binding E-box target sites. Mammalian cells transfected with CeABF-1 expression plasmids were capable of blocking E2A-mediated gene transcription, but full repression activity required the presence of two conserved amino acid residues found within the first helix of the CeABF-1 bHLH domain. These results suggest a conserved mechanism of gene repression between certain class II bHLH and class I bHLH proteins found in vertebrates and invertebrates.

A structural role for Asp83 in the photoactivation of rhodopsin.

Asp83 is a highly conserved residue in the second transmembrane domain of visual pigments and many members of other G protein-coupled receptor subfamilies. Upon illumination, the rod visual pigment rhodopsin proceeds through various intermediate states (Batho<-->BSI<-->Lumi<-->Meta I<-->Meta II). Meta II represents the active state of rhodopsin, which binds and activates the G protein transducin. Evidence has been presented that Asp83 participates in the formation of Meta II and undergoes a change in H-bonding. To investigate whether this role of Asp83 requires its proton-donating capacity and/or its H-bonding capability, we constructed the mutants D83C and D83N. Both mutants appear to effectively activate transducin, indicating that Asp83 is not essential for signal transduction. Differential effects of the mutations D83C and D83N are observed in the spectral properties and the pH sensitivity of the Meta I-->Meta II transition. In general, D83C behaves much more like wild-type than D83N. We conclude that the structural role of Asp83 also involves the acidic nature of its carboxyl group. In addition, the participation in Meta II formation of Cys83 in D83C manifests itself as a change in the vibrational properties of the sulfhydryl group, demonstrating that the -SH group can be used as a non-invasive probe for local structural changes.

Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: electronic structure of the retinylidene chromophore in rhodopsin.

11-Z-[8,9,10,11,12,13,14,15,19,20-(13)C10]Retinal prepared by total synthesis is reconstituted with opsin to form rhodopsin in the natural lipid membrane environment. The 13C shifts are assigned with magic angle spinning NMR dipolar correlation spectroscopy in a single experiment and compared with data of singly labeled retinylidene ligands in detergent-solubilized rhodopsin. The use of multispin labeling in combination with 2-D correlation spectroscopy improves the relative accuracy of the shift measurements. We have used the chemical shift data to analyze the electronic structure of the retinylidene ligand at three levels of understanding: (i) by specifying interactions between the 13C-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schiff base in rhodopsin, and (iii) by evaluating the total charge on the carbons of the retinylidene chromophore. In this way it is shown that a conjugation defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative chemical shifts at the odd-numbered carbons (Delta(sigma)odd) of 11-Z-protonated Schiff base models relative to the unprotonated Schiff base can be used to measure the extent of delocalization of positive charge into the polyene. For a series of 11-Z-protonated Schiff base models and rhodopsin, Delta(sigma)odd appears to correlate linearly with the frequency of maximum visible absorption. Since rhodopsin has the largest value of Delta(sigma)odd, the data contribute to existing and converging spectroscopic evidence for a complex counterion stabilizing the protonated Schiff base in the binding pocket.

C(2)M: configurable chemical middleware.

One of the vexing problems that besets concurrent use of multiple, heterogeneous resources is format multiplicity. C(2)M aims to equip scientists with a wrapper generator on their desktop. The wrapper generator can build wrappers, or converters that can convert data from or into different formats, from a high-level description of the formats. The language in which such a high-level description is expressed is easy enough for scientists to be able to write format descriptions at minimal cost. In C(2)M, wrappers and documentation for human reading are automatically obtained from the same user-supplied specifications. Initial experiments demonstrate that the idea can, indeed, lead to the advent of usergoverned wrapper generators. Future research will consolidate the code and extend the approach to a realistic variety of formats.

A fully functional rod visual pigment in a blind mammal. A case for adaptive functional reorganization?

In the blind subterranean mole rat Spalax ehrenbergi superspecies complete ablation of the visual image-forming capability has been accompanied by an expansion of the bilateral projection from the retina to the suprachiasmatic nucleus. We have cloned the open reading frame of a visual pigment from Spalax that shows >90% homology with mammalian rod pigments. Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (lambda(max) = 497 +/- 2 nm; pK(a) of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light). We not only provide evidence that this Spalax rod pigment is fully functional in vitro but also show that all requirements for a functional pigment are present in vivo. The physiological consequences of this unexpected finding are discussed. One attractive option is that during adaptation to a subterranean lifestyle, the visual system of this mammal has undergone mosaic reorganization, and the visual pigments have adapted to a function in circadian photoreception.

Determination of a molecular torsional angle in the metarhodopsin-I photointermediate of rhodopsin by double-quantum solid-state NMR.

We present a solid-state NMR study of metarhodopsin-1, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using double-quantum heteronuclear local field spectroscopy. The torsional angles were estimated to be [phi] = 160+/-10 degrees for rhodopsin and phi = 180+/-25 degrees for metarhodopsin-I. The result is consistent with current models of the photo-induced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.

Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.

The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.

Retinylidene ligand structure in bovine rhodopsin, metarhodopsin-I, and 10-methylrhodopsin from internuclear distance measurements using 13C-labeling and 1-D rotational resonance MAS NMR.

Rhodopsin is the G-protein coupled photoreceptor that initiates the rod phototransduction cascade in the vertebrate retina. Using specific isotope enrichment and magic angle spinning (MAS) NMR, we examine the spatial structure of the C10-C11=C12-C13-C20 motif in the native retinylidene chromophore, its 10-methyl analogue, and the predischarge photoproduct metarhodopsin-I. For the rhodopsin study 11-Z-[10,20-(13)C(2)]- and 11-Z-[11,20-(13)C(2)]-retinal were synthesized and incorporated into bovine opsin while maintaining a natural lipid environment. The ligand is covalently bound to Lys(296) in the photoreceptor. The C10-C20 and C11-C20 distances were measured using a novel 1-D CP/MAS NMR rotational resonance experimental procedure that was specifically developed for the purpose of these measurements [Verdegem, P. J. E., Helmle, M., Lugtenburg, J., and de Groot, H. J. M. (1997) J. Am. Chem. Soc. 119, 169]. We obtain r(10,20) = 0.304 +/- 0.015 nm and r(11,20) = 0.293 +/- 0.015 nm, which confirms that the retinylidene is 11-Z and shows that the C10-C13 unit is conformationally twisted. The corresponding torsional angle is about 44 degrees as indicated by Car-Parrinello modeling studies. To increase the nonplanarity in the chromophore, 11-Z-[10,20-(13)C(2)]-10-methylretinal and 11-Z-[(10-CH(3)), 13-(13)C(2)]-10-methylretinal were prepared and incorporated in opsin. For the resulting analogue pigment r(10,20) = 0.347 +/- 0.015 nm and r((10)(-)(CH)()3())(,)(13) = 0.314 +/- 0.015 nm were obtained, consistent with a more distorted chromophore. The analogue data are in agreement with the induced fit principle for the interaction of opsin with modified retinal chromophores. Finally, we determined the intraligand distances r(10,20) and r(11,20) also for the photoproduct metarhodopsin-I, which has a relaxed all-E structure. The results (r(10,20) >/= 0.435 nm and r(11,20) = 0.283 +/- 0.015 nm) fully agree with such a relaxed all-E structure, which further validates the 1-D rotational resonance technique for measuring intraligand distances and probing ligand structure. As far as we are aware, these results represent the first highly precise distance determinations in a ligand at the active site of a membrane protein. Overall, the MAS NMR data indicate a tight binding pocket, well defined to bind specifically only one enantiomer out of four possibilities and providing a steric complement to the chromophore in an ultrafast ( approximately 200 fs) isomerization process.

Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system.

Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni(2+)-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin.

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [alpha,epsilon-15N2]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [alpha, epsilon-15N2]Lys-rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [alpha, epsilon-15N2]Lys-rhodopsin resonates with an isotropic shift sigmaI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, numax, associated with the pi-pi transition of the polyene chromophore. Experimental modeling of the relation between the numax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center-center distance (deff) between the counterion and the pSB of 0.43 +/- 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.

Tyrosine structural changes detected during the photoactivation of rhodopsin.

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho --> MII difference spectra were obtained at 10 degrees C for unlabeled recombinant Rho and isotope-labeled L-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2H4]Tyr and isolated using a His6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR --> M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the F-helix, may form a hinge that facilitates alpha-helix movement.

Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates.

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes.

A novel generic approach is described for the selective extraction of detergents from mixed detergent/lipid/protein micelles for the preparation of proteoliposomes of defined lipid-protein ratio. The approach is based on the much higher affinity of inclusion compounds of the cyclodextrin type for detergents in comparison with bilayer-forming lipids. This approach has distinct advantages over other procedures currently in use. It produces good results with all detergents tested, independent of type and critical micelle concentration, and appears to be generally applicable. It yields nearly quantitative recovery of membrane protein in the proteoliposome fraction. Finally, no large excess of lipid is required; a molar ratio of lipid to protein of 100 to 1 already produces proteoliposomes with functional membrane protein, but higher ratios are well tolerated. The size of the vesicles thus obtained depends on the detergent used. Separation of the resulting proteoliposomes from the detergent-cyclodextrin complexes was most easily achieved by centrifugation through a discontinuous sucrose gradient. A variety of detergents was tested in this procedure on the bovine rod visual pigment rhodopsin in combination with retina lipids. In all cases good yields of proteoliposomes were obtained, which contained fully functional rhodopsin.

An additional methyl group at the 10-position of retinal dramatically slows down the kinetics of the rhodopsin photocascade.

The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.

Steady-state pharmacokinetics of oestradiol gel in post-menopausal women: effects of application area and washing.

OBJECTIVE: To investigate the effect of cutaneous application area on oestradiol absorption using an oestradiol gel for transdermal use. Furthermore, the effect of washing of the application site on oestradiol pharmacokinetics was studied. DESIGN: Open-label, randomised, three-way cross-over study. SETTING: A clinical pharmacokinetic research unit. SUBJECTS: Sixteen healthy postmenopausal volunteers. INTERVENTIONS: During three treatment periods, subjects were treated with 1 mg oestradiol (1.0 g Divigel/Sandrena 0.1% gel), applied on the thigh to a skin area of 200 cm2, 400 cm2, or on an area 'as large as possible'. Blood samples were drawn during steady-state (on days 14-15) immediately before application and at regular time intervals thereafter. MAIN OUTCOME MEASURES: Serum oestradiol levels were measured for determination of peak concentration (Cmax), time to peak concentration (tmax) and bioavailability using the area under the time concentration curve (AUC0-24). RESULTS: Bioavailability from a 200 cm2 area was 2-fold higher than that from the largest possible area (P < 0.05). Oestradiol peak plasma concentration from a 200 cm2 area was higher than from the two larger areas (P < 0.05), which did not differ significantly from each other. Washing of the application site 30 min after application reduced the bioavailability from both the 200 cm2 and 400 cm2 application areas (P < 0.01), but peak plasma concentration from the 200 cm2 area only (P < 0.01). Time to reach peak plasma concentrations was significantly reduced with all three application areas by washing. CONCLUSION: A higher oestradiol absorption was achieved from a smaller application area. No marked differences were observed in the pharmacokinetics between 200 cm2 and 400 cm2 application areas, which are those recommended for this oestradiol gel preparation.

Transdermal oestradiol gel in the treatment of the climacterium: a comparison with oral therapy.

OBJECTIVE: To compare two doses of a transdermal oestradiol gel (Divigel/Sandrena) plus oral sequential medroxyprogesterone acetate (MPA) with oral oestradiol valerate plus oral sequential MPA (Divina/Dilena). DESIGN: Two year, randomised, open-label, comparative study. SETTING: Menopausal outpatient clinic in Helsinki. SUBJECTS: Postmenopausal women with climacteric complaints or already using HRT. INTERVENTIONS: (1) One gram gel containing 1 mg oestradiol for 3 months plus 20 mg oral MPA during the last 14 days; (2) 2 g gel containing 2 mg oestradiol for 21 days plus 10 mg oral MPA during the last 14 days; (3) 2 mg oestradiol valerate tablets for 3 weeks plus 10 mg oral MPA during the last 10 days. In all groups, each treatment period was followed by a 7-day medication-free interval. MAIN OUTCOME MEASURES: Climacteric complaints, bleeding control, bone mineral density, biomarkers of bone metabolism, lipid profile, tolerability and safety. RESULTS: With each preparation, climacteric complaints were significantly reduced and good bleeding control was obtained. In addition, maintenance of bone mineral density as well as a reduction of bone turnover was achieved in all groups. Lipid parameters showed no unfavourable changes. Continuation rates were similar in all groups with overall 74% of patients completing the first year, whereas 94% of patients who elected to continue completed the second year. Tolerability of the gel was good: only 1.7% of patients discontinued treatment due to skin irritation. CONCLUSIONS: Transdermal oestradiol gel and oral oestradiol valerate tablets, used in combination with oral sequential MPA, are effective regimens of HRT in postmenopausal women. Transdermal oestradiol gel is an efficient, well-tolerated form of HRT.