Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.

The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.

[Non-oral administration of progesterone: experiences and possibilities of the transvaginal route]. / Administration non orale de la progestérone: expériences et avenir de la voie transvaginale.

Recent development of transdermal therapy permits application of estrogen, usually produced in the ovaries, in physiological dosage by means of continuous release from either an epidermal patch or dermal application of a gel. Transdermal therapy with progesterone, however is impossible due to poor dermal absorption and high dose requirements (release from corpora lutea: 25 mg/24 hours). Two other possibilities have been proposed. On one hand it is possible to apply norethisterone-acetate (NETA), another gestagen, epicutaneously. This mode of administration carries the same problems as oral application thus allowing for a dose reduction. On the other hand progesterone can be applied vaginally. This mode leads to significantly higher plasmatic concentrations of progesterone and has effects on the uterine mucosa similar to those in a normal cycle. This modality thus permits application of estradiol and progesterone in a physiological manner by a non oral route. It appears particularly interesting in patients at cardiovascular risk.

Mutation of conserved domain II alters the sequence specificity of DNA binding by the p53 protein.

We have mutagenized human p53 expressed in yeast and selected two mutants, 121F and 123A, which activate transcription from one, rather than the normal two, copies of the consensus p53 DNA binding sequence. Both mutants have a 6-fold increase in affinity for a single copy of the sequence GGG CATG CCC. The 121F mutant has a decrease, and the 123A mutant an increase, in the affinity for the sequence GAA CATG TTC. This genetic and biochemical evidence supports the crystallographic finding that amino acid 120 contacts guanine in the major groove at the second position in the consensus. The major p53 binding site in the p21WAF1/CIP1 promoter resembles the GAA CATG TTC form of the consensus. Compared with wild type p53, the 121F mutant has a 7-fold lower affinity for the p21WAF1/CIP1 site in vitro, and the 121F mutant is defective in p21 induction in vivo. Mutants with subtly altered sequence specificity may facilitate dissection of downstream pathways activated by p53.

Mammalian p53 can function as a transcription factor in yeast.

p53 has previously been shown to contain a transactivation domain using GAL4 fusion proteins and to bind specifically to a 33 base pair DNA sequence in immunoprecipitation assays. We show here that mammalian p53 expressed in S. cerevisiae is able to activate transcription of a reporter gene placed under the control of a CYC1 hybrid promoter containing the 33 base pair p53-binding sequence. The activation is dependent on the orientation and number of copies of the binding site. Three p53 mutants commonly found in human tumours, 175H, 248W and 273H, are unable to activate transcription. A fourth human p53 mutant, 285K, is temperature-sensitive for transcriptional activation. Murine p53 activates transcription from the same sequence. The murine 135V mutant, which is temperature-sensitive for mammalian cell transformation, is also temperature-sensitive for transcriptional activation. There is a much better correlation between mutation and transcriptional competence than between mutation and the structure of p53 determined with conformation-sensitive antibodies. We have therefore developed a simple transcription assay for p53 mutation in which yeast are transfected with p53 PCR products and mutation is scored on X-gal plates.

Predictive value of pharmaco-electroencephalography in early human-pharmacological evaluations of psychoactive drugs. First example: savoxepine.

The present paper forms the first part of a contribution to a comprehensive critical evaluation of the use of pharmaco-electroencephalography (pharmaco-EEG) as an instrument in the evaluation of pharmacodynamic effects of psychoactive drugs in man and further prediction of therapeutic effects by utilizing partial effects in a clinical pharmacological model situation. The basic principles and the methodological approaches are described first, prior to discussing the possibilities and limitations of the pharmaco-EEG in postulating hypotheses about therapeutic efficacy of psychoactive drugs in man. The discussion rests upon the findings with three selected new psychoactive drugs (savoxepine, levoprotiline and maroxepine), presented in this (Part I) and subsequent (Part II and III) reports. In this report results obtained with savoxepine, a novel tetracyclic compound, are described. Convergent evidence for antipsychotic potential of this drug has emerged from preclinical and pharmaco-EEG studies, subsequently corroborated by the first open trials in patients suffering from acute episodes of schizophrenia. In the pharmaco-EEG study a single dose of 0.5 mg savoxepine was tested in 15 healthy subjects with comparison to chlorpromazine (75 mg) and placebo. The experimental design was a threefold cross-over uncompleted block design with three consecutive trial days one week apart. Pharmaco-EEG was recorded under a high situational level of vigilance before 3 h and 6 h after drug administration. The recording was performed by means of 12 electrodes placed in the frontal, central, parietal and occipital regions. EEG-maps were constructed after power spectral analysis of absolute power changes in predetermined frequency bands within the range 0.5 to 30.0 Hz. The findings indicated EEG effects of savoxepine similar to those of chlorpromazine. In the tested dosage, however, savoxepine showed higher potency, later maximum (6 h) of the effect and lower sedative potential. Clinical trials performed in two centres and in, altogether, 28 acutely psychotic patients indicated antipsychotic activity of the drug in a low range of doses (on average, 0.1 to 0.9 mg/d). Even though the antipsychotic efficacy of savoxepine remains to be confirmed in controlled studies, the results favour the hypothesis that pharmaco-EEG has a value in predicting the quality of action of a new drug in man. Results also favour the complementary use of pharmaco-EEG in human pharmacology testing in order to objectively assess central effects of drugs and rationally estimate the doses for therapeutic trials.

Pharmaco-EEG profile of levoprotiline: second example to discuss the predictive value of pharmaco-electroencephalography in early human pharmacological evaluations of psychoactive drugs.

The present paper forms the second part of a critical evaluation of the use of pharmaco-EEG as an instrument for assessing the profile of action of psychoactive drugs in man based on the trial results of three new psychoactive test substances. Part I described the basic principles and methodological approaches and illustrated the value of pharmaco-EEG in framing hypotheses about the therapeutic efficacy of psychotropic drugs by the example of the neuroleptic drug savoxepine. This chapter illustrates the relevance of pharmaco-EEG in the assessment of psychoactive drugs by reference to the test substance levoprotiline. Levoprotiline is the pure R-(-)-enantiomer of the racemic drug oxaprotiline, a successor to the second-generation antidepressant maprotiline. Only the S-(+)-enantiomer of oxaprotiline inhibits the re-uptake of noradrenaline. In view of the absence of any monoamine-uptake inhibiting action of levoprotiline, this drug was assumed to be devoid of an antidepressive action. This assumption, however, was disproved by observations made in clinical studies with depressive patients, in whom levoprotiline did indeed display antidepressive activity. Investigations of levoprotiline in the pharmaco-EEG model--even though being retrospective in the sequence of events--would have been able to produce objective prediction of antidepressive potential in man. The substance was tested by comparison to placebo and to the standard antidepressant imipramine in nine young, healthy male volunteers. The experimental design was a cross-over uncompleted block design with three consecutive trial days one week apart.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmaco-EEG profile of maroxepine: third example to discuss the predictive value of pharmaco-electroencephalography in early human pharmacological evaluations of psychoactive drugs.

This publication forms the third part of a critical evaluation of pharmaco-EEG applications as an instrument for assessing the profile of action of psychoactive drugs based on the trial results of three new psychoactive test substances: savoxepine, levoprotiline and maroxepine. After the presentation, in Parts I (savoxepine) and II (levoprotiline), of trial substances for which compatible results were obtained in the pharmaco-EEG model and in the clinical studies, a case with discrepant and less predictive results has been presented, and on this basis the limitations of the pharmaco-EEG model in the evaluation of therapeutic efficacy are discussed. The substance examined in this report, the tetracyclic dibenzoxepine derivative, maroxepine, produced prominent central effects in pharmacological studies which pointed to a bipolar profile of action, i.e., the EEG showed similarities both to antidepressants and antipsychotics. The pharmaco-EEG effects of maroxepine were investigated in a randomised, double-blind study in 15 young healthy male subjects and compared with those of placebo and the reference substances, chlorpromazine and imipramine. The experimental design was a cross-over uncompleted block design with five consecutive trial days one week apart. The pharmaco-EEG was recorded from the occipito-temporal and frontocentral regions before, 3 and 6 h after application of 2.5 mg and 5.0 mg maroxepine, 75 mg chlorpromazine, 75 mg imipramine and placebo. The selected target variables for descriptive statistical evaluation were the Spectral Difference Index (SDI), the absolute and relative power values in seven predetermined frequency bands within the range 1.5 to 30.0 Hz, the dominant frequency (6.0 to 18.0 Hz) and the alpha slow-wave index (ASI). The results show that maroxepine has a potent central nervous action and a strong sedative potential. The EEG effects consisted of a shift to the left of the relative power spectrum of the occipital lead, accompanied by an increase in the absolute beta power in the frontocentral region. Discriminative inspection of the profiles of EEG changes, induced by maroxepine and the reference drugs, revealed closer similarity of maroxepine with chlorpromazine than with imipramine. Maroxepine showed vigilance-decreasing features like imipramine. This is one condition to be able to interfere with depressive illness. The underlying hypothesis is that depressive illness is based on affective and vigilance disturbance. Maroxepine furthermore showed the typical neuroleptic-like shift from resting alpha to resting subalpha (theta F band) which we assume related to the anti-productive properties of neuroleptics in schizophrenia.(ABSTRACT TRUNCATED AT 400 WORDS)

The long-term tolerability of bencyclane ('Fludilat') in patients with peripheral occlusive disease: a 48-week prospective double-blind controlled study versus placebo.

In a controlled, multi-centre, double-blind trial, 75 patients with Stage II peripheral occlusive disease (Fontaine IIa) were treated with either 200 mg bencyclane twice daily or placebo over a period of 12 months. Undesired drug effects and concomitant phenomena were documented, and efficacy was evaluated. Bencyclane caused a slight, clinically negligible decrease in blood pressure. The pulse rate remained mostly unchanged, ECG and laboratory parameters showed no changes which would indicate a specific effect of the test substance. In the context of the generally low incidence of concomitant effects, patients in the bencyclane group mentioned symptoms such as insomnia, depressive mood, sweating and reduced motoricity more often than those in the placebo group. These symptoms are regarded as signs of the central nervous actions of the drug. The parameters used to assess the efficacy, i.e. the pain-free walking distance estimated by the patients and the physician's global judgment based on Ratschow's test, the palpability of the pedal pulse, the walking range and the patients' subjective statements about the incidence of chill, formication, and pain in the legs, showed a constant and statistically significant superiority of bencyclane over placebo.

Two-dimensional DNA-fingerprinting in animals.

DNA-fingerprinting has become, during the last five years, an important method of genetic analysis in medicine, veterinary medicine and other disciplines. The power of this technique, especially for genetic linkage analysis, may be enhanced in humans by using the two dimensional DNA-fingerprinting method. Here we show that this procedure can successfully be applied to different animal species, e.g. pig, dog and mouse. Optimal conditions, however, have to be determined for each species tested. With the use of marker systems as well as computer programs it will be possible to evaluate complex two-dimensional spot patterns in a short time and with high reliability.