A design for the perfusion of one (monoperfusion) or two (parallelperfusion) cotyledos of one placenta was developed for studies of the metabolism of the precursor steroid dehydroepiandrosterone (DHA). Several parameters are used as viability criteria: vascular resistance, glucose and oxygen consumption, lactate/pyruvate ratio, activity of lactate-dehydrogenase (LDH) in the perfusate, extent of perfusion by dye infusion, and morphological description by electron microscopy. A dosage of 2 mg DHA with 2.5 microCi 14C-radioactive labelled marker is given for testing the metabolizing capacity of the placenta. The labelled metabolites DHA, androstenedione (A), testosterone (T), estrone (Oe 1), and estradiol-17 beta (Oe 2) are separated by thin-layer chromatography and measured by scanning and measurements of scraped radioactive spots by scintillation counting. The steroidogenesis is evaluated with the concentrations of Oe 1 at 15 min (Oe 1.15'), Oe 1 + Oe 2 at 90 min (Oe tot, 90'), total aromatization rate (from perfusate and homogenate after 120 min). Results comparable to DHA are found using DHA-sulphate (DHA-S) as precursor, higher amounts of estrogens are metabolized from A. Mature placentas metabolize DHA in relation to the initial DHA concentration: optimal aromatization is found at 250-350 pmol DHA/ml/g, decreased aromatization at higher or lower concentrations. Oe 1 represents the main placental metabolite.

Androstenedione↗

PubMedDate not supplied

BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli.

A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.

Amino Acid Sequence↗

PubMedDate not supplied

[Biology of female sexuality in old age].

In the aged women, beginning after the menopause, sexuality will be declining in a high percentage. The reasons are many: bad state of health, complaints, decrease of fitness, genital atrophy, gynecological operations, partner problems, and others. On the other side, sexuality can be maintained up to high age. In any case is the demand for affection, tenderness, love and sexual relations nearly always present, and its realization should not be inhibited. The sexual reactions are decreased and retarded. Therefore often a change of the role and behaviour pattern of the partners is necessary in the sexual contact. The partners should know their mutual problems and desires, show understanding and should adjust to each other. Counselling, behaviour therapy and treatment with sexual hormones can help to solve the problems.

Aged↗