Comparative effects of chlormadinone acetate and its 3alpha- and 3beta-hydroxy metabolites on progesterone, androgen and glucocorticoid receptors.

AIM/METHODS: In vitro binding tests to human receptors and in vivo functional activities in animals were used to compare the effects of the progestin chlormadinone acetate (CMA) and its 3alpha- and 3beta-hydroxy metabolites (3alpha-OH-CMA and 3beta-OH-CMA) on progesterone, androgen and glucocorticoid receptors. RESULTS: CMA, 3alpha-OH-CMA, 3beta-OH-CMA and the reference progestin R5020 bound to human progesterone receptor with Ki values of 2.5 nm, 13 nm, 6.0 nm and 4.3 nm, respectively. Binding affinities to the human androgen receptor were characterized by Ki values of 3.8 nM for CMA, 83 nM for 3alpha-OH-CMA, 20 nM for 3beta-OH-CMA and 2.9 nM for the reference androgen methyltrienolone. The Ki values for binding to the human glucocorticoid receptor were 16 nM for CMA, 69 nM for 3alpha-OH-CMA, 21 nM for 3beta-OH-CMA and 1.2 nM for the glucocorticoid dexamethasone. In the rabbit endometrial proliferation test CMA, 3alpha-OH-CMA and 3beta-OH-CMA (5 and 45 microg/kg p.o. for 5 days) had similar progestomimetic activities. CMA, 3alpha-OH-CMA and, to a lesser extent, 3beta-OH-CMA (4.64 and 21.5 mg/kg p.o. for 7 days) inhibited testosterone-stimulated growth of prostate and seminal vesicles in castrated rats showing antiandrogenic activities. Glucocorticoid properties were demonstrated for CMA and 3alpha-OH-CMA (21.5 and 100 mg/kg p.o. for 6 days) but not for 3beta-OH-CMA as reduction in thymus and adrenal gland weights in immature rats. CONCLUSION: Binding assays at human receptors showed similarly high affinities of CMA with the progesterone and androgen receptors and a 5 times lower affinity with the glucocorticoid receptor. At all receptor types, CMA had the highest, 3alpha-OH-CMA the lowest and 3beta-OH-CMA an intermediate affinity. Animal studies revealed progestomimetic and antiandrogenic activities of CMA, 3alpha-OH-CMA and 3beta-OH-CMA and glucocorticoid activities of CMA and 3alpha-OH-CMA.

Resonance Raman spectroscopic study of the tryptic 39-kDa fragment of phytochrome.

The 39-kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state P(r) reveals far-reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the P(r) phototransformation, denoted as P(bl), is more closely related to that of the P(fr) precursor of the native phytochrome, i.e. meta-R(C), rather than to that of P(fr) itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in P(bl) so that, unlike in P(fr), the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of P(fr), cannot be established in the 39-kDa fragment due to the lack of the major C-terminal part of the protein. These findings, furthermore, support the view that the meta-R(C)-->P(fr) transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome.

Effect of chromophore exchange on the resonance Raman spectra of recombinant phytochromes.

The recombinant 65-kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (PphiB) and phycocyanobilin (PCB), respectively. The Pr and Pfr states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA oat phytochrome demonstrating that the deletion of the C-terminal half of phyA does not alter the structure of the chromophore site within the N-terminal half. Most of the RR spectral changes observed upon replacing PphiB by PCB can be attributed exclusively to altered vibrational mode compositions due to the different ring D substitutions (vinyl vs. ethyl), implying that the chromophore structures are largely the same for PphiB- and PCB-assembled phytochromes. Only in the Pr state may the RR spectral changes also reflect subtle differences of the PphiB and PCB conformations in the 65-kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups.

Influences of gold on zinc, copper and metallothionein kinetics in liver and kidney of the rat.

1. Single, very low dose of gold (500 micrograms/kg body weight) was given intramuscularly to male Sprague-Dawley rats (mean body weight: 200 g) as aurothiokeratinate (Auro-Detoxin(R) dissolved in 0.9% NaCl. 2. Blood, liver and kidney were samples at autopsy 0.5, 1, 2, 3, 6 and 12 h after the gold injection (six animals per time). 12 rats were treated with 0.9% saline only as controls. The zinc (Zn), copper (Cu), gold and metallothionein (MT) concentrations were determined in homogenates and cytosols of liver and kidney using atomic absorption spectrometry and gel chromatography, respectively. 3. The main changes in metal and MT concentrations occurred in the kidney, where an increase of gold was found 0.5 h after the injection, followed by an increase in Cu and MT concentrations after 6 h. Zn homeostasis did not change. 4. The results suggest that gold by itself induces an increase of MT-like peptides in the kidney cytosol, which was accompanied by an increase in the concentration of Cu mainly bound to these MT-like peptides.

Protonation state and structural changes of the tetrapyrrole chromophore during the Pr --> Pfr phototransformation of phytochrome: a resonance Raman spectroscopic study.

The photoconversion of phytochrome (phytochrome A from Avena satina) from the inactive (Pr) to the physiologically active form (Pfr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic temperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D exchange on the resonance Raman spectra. For the first time, reliable spectra of the "bleached" intermediates meta-R(A) and meta-R(C) were obtained. The vibrational bands in the region 1300-1700 cm(-)(1), which is particularly indicative of structural changes in tetrapyrroles, were assigned on the basis of recent calculations of the Raman spectra of the chromophore in C-phycocyanin and model compounds [Kneip, C., Hildebrandt, P., Németh, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental resonance Raman spectra Pr are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the chromophore structure in this parent state of phytochrome. Furthermore, marker bands could be identified that are of high diagnostic value for monitoring structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states Pr and Pfr but also in all intermediates the chromophore is protonated at the pyrroleninic nitrogen. The spectral changes observed for lumi-R confirm the view that the photoreaction of Pr is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-R(A) includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R(C) is accompanied by structural adaptations of the pyrrole rings B and C in the protein pocket. The far-reaching similarities between the chromophores of meta-R(A) and Pfr suggest that in the step meta-R(A) --> Pfr the ultimate structural changes of the protein matrix occur.

Raman spectroscopic and light-induced kinetic characterization of a recombinant phytochrome of the cyanobacterium Synechocystis.

A phytochrome-encoding cDNA from the cyanobacterium Synechocystis has been heterologously expressed in Escherichia coli and reconstituted into functional chromoproteins by incubation with either phycocyanobilin (PCB) or phytochromobilin (PPhiB). These materials were studied by Raman spectroscopy and nanosecond flash photolysis. The Raman spectra suggest far-reaching similarities in chromophore configuration and conformation between the Pfr forms of Synechocystis phytochrome and the plant phytochromes (e.g. phyA from oat), but some differences, such as torsions around methine bridges and in hydrogen bonding interactions, in the Pr state. Synechocystis phytochrome (PCB) undergoes a multistep photoconversion reminiscent of the phyA Pr --> Pfr transformation but with different kinetics. The first process resolved is the decay of an intermediate with red-shifted absorption (relative to parent state) and a 25-micros lifetime. The next observable intermediate grows in with 300 (+/-25) micros and decays with 6-8 ms. The final state (Pfr) is formed biexponentially (450 ms, 1 s). When reconstituted with PPhiB, the first decay of this Synechocystis phytochrome is biexponential (5 and 25 micros). The growth of the second intermediate is slower (750 micros) than that in the PCB adduct whereas the decays of both species are similar. The formation of the Pfr form required fitting with three components (350 ms, 2.5 s, and 11 s). H/D Exchange in Synechocystis phytochrome (PCB) delays, by an isotope effect of 2.7, both growth (300 micros) and decay rates (6-8 ms) of the second intermediate. This effect is larger than values determined for phyA (ca. 1.2) and is characteristic of a rate-limiting proton transfer. The formation of the Pfr state of the PCB adduct of Synechocystis phytochrome shows a deuterium effect similar as phyA (ca. 1.2). Activation energies of the second intermediate in the range 0-18 degrees C are 44 (in H2O/buffer) and 48 kJ mol-1 (D2O), with essentially identical pre-exponential factors.

[Tuberculous spondylitis--a forgotten differential diagnosis in backache]. / Spondylitis tuberculosa--eine vergessene Differentialdiagnose bei Rückenschmerzen.

A four-year-old boy had been complaining for one year of backache when sitting or bending. Radiological changes in the lumbar spine were at first interpreted as due to an old vertebral fracture. Half a year after the onset of the symptoms his sister was found to have open pulmonary tuberculosis, but a positive Tine test (he had not received BCG vaccination) was without any consequences. He was finally hospitalized five months later because the backache persisted and he now also had impaired movement of the left hip-joint. Physical examination revealed a 10 x 15 cm firm mass in the abdomen. Imaging demonstrated destruction of the 4th and 5th lumbar vertebrae and a large abscess. Acid-fast bacteria were found in gastric juice. Tuberculostatic treatment was started with isoniazid (10 mg/kg), rifampicin (10 mg/kg) and pyrazinamide (30 mg/kg). Two weeks later spondylectomy was performed, the defect being bridged with bone chips from the iliac crest. Four months after the operation, tuberculostatic treatment now being only isoniazid and rifampicin, he was able to partake fully in all activities of his age group.