Effect of curl-free potentials on water.

Living objects are complex systems with various harmonized chemical, thermodynamical, and quantum-mechanical processes in aqueous electrolyte environment. We had studied the effect of curl-free magnetic vector-potential on the matrix of the living matter, on the water. The discussed theoretical considerations are in harmony with the presented simple experiments. It is shown that the vector-potential is actually an effective electro-dynamical parameter which could modify the processes in living systems.

Pink-noise behaviour of biosystems.

Pink (1/f) noise is one of the most common behaviours of biosystems. Our present paper is devoted to clarify the origin of this interesting phenomenon. It is shown that the stationary random stochastic processes under self-similar conditions (as we have in living objects) generate pink noise independently of the kind and number of variables.

Evidence for a role of Smad6 in chick cardiac development.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are obligatory growth factors for early embryogenesis and heart formation. SMAD proteins transduce signals of the TGF-beta superfamily. We isolated chicken Smad6 (cSmad6), a member of inhibitory SMADs, and found its expression to be remarkably restricted to the developing heart, eyes, and limbs. cSmad6 expression was detected in the cardiogenic region of stage 5 embryos and overlapped Nkx2-5 and bmp-2, -4, and -7 expression. Throughout development, cSmad6 was expressed strongly in the heart, primarily in the myocardium, endocardium, and endocardial cushion tissue. Myocardial expression of cSmad6 was stronger in the forming septum, where highly localized expression of bmp-2 and -4 was also observed. Ectopically applied BMP-2 protein induced the expression of cSmad6, a putative negative regulator of BMP-signaling pathway, in anterior medial mesoendoderm of stage 4-5 embryos. In addition, blocking of BMP signaling using Noggin downregulated cSmad6 in cardiogenic tissue. cSmad1, one of the positive mediators of BMP signaling, was also expressed in cardiogenic region, but was not BMP-2 inducible. Our data suggest that cSmad6 has a role in orchestrating BMP-mediated cardiac development. We propose the possible mechanism of action of cSmad6 as modulating BMP signal by keeping a balance between constitutively expressed pathway-specific cSmad1 and ligand-induced inhibitory cSmad6 in the developing heart.

Alterations in the subcellular distribution of 17 beta-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle.

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17 beta-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2-4 microns diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.

Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Comparison of the retention of receptor in nuclei during subcellular fractionation of untreated and hormone-treated cells.

Nuclei were isolated from porcine endometrium of castrated pigs either unexposed or exposed to estradiol in vivo by two techniques, one of which included a hypotonic step. Aliquots were analyzed for estradiol content. Receptor was extracted from buffered, Surfynol-stabilized suspensions by either (a) KCl alone, (b) in combination with dithiothreitol, or (c) by dithiothreitol with polypentosanesulfate and addition of KCl. The yields rose from a-->c. The same proportional gains with increasing extractant efficacies were obtained from nuclei of unstimulated and estradiol-treated cells. Receptor recovery with extractant "c" rose linearly over the range of 9-80 x 10(6) nuclei/mL and was independent of the technique used for isolation. Nuclear fractions isolated using steroid-free solutions contained more estrogen receptor than estradiol; the numerical excess in control nuclei persisted in the nuclei of stimulated cells featuring a stoichiometric rise of ligand and receptor contents. The increase of receptor contents in nuclei isolated from hormone-stimulated cells coincided with a decline in the cytoplasmic fractions. An excess of hormone over receptor was seen only when nuclei were isolated from untreated cells with media containing 10 nM estradiol. Our data strengthen earlier notions of an estradiol-promoted receptor translocation into the nucleus and are not compatible with the ligand-filling hypothesis of preexisting nuclear binding sites.

Biosynthesis and posttranslational finishing of the estradiol receptor.

The time course of subcellular receptor distribution in porcine endometrial epithelium was studied after intrauterine administration of estradiol alone or in combination with puromycin. In untreated cells, the major proportion of receptor is associated with cytoplasmic membranes. The solubilization of receptor from isolated nuclei is independent of their estradiol content. Smooth cytoplasmic membranes are the site of origin of receptor which is swiftly translocated into the nucleus in a 1:1 ratio with the hormone after exposure of the cells to estradiol. Simultaneously administered puromycin delays receptor synthesis and reveals that the nuclear passage of receptor is terminated by receptor degradation. The synthesis of receptor proceeds in rough endoplasmic membranes. A subsequent finishing and deposition in smooth membranes depends on intact protein synthesis.

Origin and quantification of cytoplasmic estradiol receptor in resting target cells.

The exhaustive extraction of microsomal estradiol receptor by Surfynol/dithiothreitol/trypsin in low ionic strength buffer was employed for distribution studies on non-stimulated porcine endometrium. It was found that more than half of the cytoplasmic receptor contents were of microsomal origin. "Empty" structures did not interfere with receptor analysis by agargel electrophoresis. The combined yields from homogenate fractions corresponded to those obtained from unfractionated homogenates. Freeze-fracturing of endometrium had a moderate receptor-solubilizing effect.

Structural assignment and extractability of microsomal estradiol receptors.

Two electrophoretically different forms of estradiol receptor can be extracted from crude porcine endometrium microsomes with low ionic strength buffers. Better yields (approximately 50%) of both forms are obtained in the presence of Surfynol 485. Dithiothreitol boosts the solubilization of basic receptor. Together, Surfynol and dithiothreitol have a more than additive effect, amounting to 3-4 times the quantities of receptor extracted with plain buffer. Trypsin more than triples the yields obtained with Surfynol/dithiothreitol, while degrading both receptor forms to a characteristic fragment. Hyaluronoglucosaminidase is somewhat less effective than trypsin. It changes acidic receptor to basic. The proportions of acidic/basic receptor in microsomal subfractions are different. Rough endoplasmic reticulum contains almost exclusively basic receptor. Smooth membranes are rich in acidic receptor. The efficacy of both enzymes is closely related to the proportion of acidic receptor found in Surfynol/dithiothreitol extracts.

Characterization of microsomal subfractions from porcine endometrium cells.

Crude microsomes from porcine endometrium and three subfractions obtained by a modification of Rothschild's technique were characterized by RNA/protein ratio, marker enzyme activities and morphological appearance. The microsomes were devoid of glucose-6-phosphatase activity. They contained approximately 10% of arylesterase-, approximately 30% of both NADPH-cytochrome reductase- and UDPgalactose-N-acetyl-glucosamine beta-D-galactosyltransferase- and approximately 60% of 5'-nucleotidase activities present in the homogenates. Subfraction I (smooth membranes) had twice the galactosyltransferase activity of Subfraction II (smooth and rough membranes + free ribosomes); both subfractions were rich in 5'-nucleotidase and cytochrome reductase activities. Subfraction III (rough membranes) had very low marker activities but exhibited the highest RNA/protein ratio, which was lowest in I.

Subcellular distribution, properties and interrelationship of oestrogen receptors in endometrium and other target tissues.

More than half of the extranuclear receptor content of resting cells is associated with cytoplasmic structures. Subfractionation of microsomes reveals a preponderance of "basic" (low electrophoretic mobility) receptor in rough endoplasmic reticulum. Surfynol-dithiothreitol extracts of smooth membranes are rich in "acidic" (high electrophoretic mobility) receptor. Trypsin increases the yields up to seven-fold, and this increase is correlated (r = 0.993) with the acidic receptor content and 5'-nucleotidase activity of these microsomal preparations. Acidic microsomal "cytosolic" and nuclear receptor can be degraded to the "basic" variety of streptomyces hyaluronidase. All forms give rise to a tryptic fragment with unchanged affinity for oestradiol and dimerization ability. Basic receptor isolated after enzymatic conversion of acidic receptor is microheterogenous on isoelectric focusing, but gives rise to only one precipitation arc versus the IgG fraction of a polyclonal antiserum. The precipitation arc can be recharged with labelled oestradiol and autoradiographed. Non-immune IgG's from (unspecific) soluble complexes with oestrogen receptors, but not with their tryptic fragments. The polyclonal antioestrogen receptor IgG fraction precipitates the oestradiol-tagged tryptic receptor fragment from all subcellular sources of all homologous (porcine) and heterologous (human, ovine, bovine, goat, rabbit, guinea pig, rat) target tissues tested. Organ specificity can therefore be excluded and a high degree of phylogenetic conservation of the oestrogen receptor's protein moiety is implied. The presence, in the immune IgG fraction, of steroid releasing antibodies, which apparently distort the binding site, should spur the search for monoclonal antibodies with similar properties.

2. Mechanism of steroid action. Immunoreactivity of the core of estrogen receptors found in different subcellular compartments of target cells from a variety of mammalian species.

A goat antiserum was raised by immunization with the endoglycosidase fission product of porcine estrogen receptor. Its IgG fraction was employed for ascertaining the interrelationship of receptor forms extracted from three subcellular compartments of target cells and for comparing receptors from various species. Unspecific alignments of (non-immune) IgGs giving rise to soluble complexes were avoided by removal of the receptor entity responsible with carrier-attached trypsin. Coincubation of the estradiol-tagged tryptic receptor cores and immune IgG resulted in the formation of labelled precipitates and in the release of estradiol from its binding site, in every instance tested. A minimum of three common antigenic determinants must therefore exist not only on all porcine receptor forms, but also on human, ovine, bovine, rabbit, guinea-pig and rat estrogen receptors, pointing to a high degree of phylogenetic conservation.

Validation of the common-core hypothesis of estrogen receptors with precipitating and steroid-releasing antibodies.

The immunoglobulin G fraction of a goat antiserum raised against a presumed endoglycosidase-fission product of estradiol receptor from porcine uteri contains some antibodies which precipitate the estradiol/receptor complex and others which release the steroid from the protein. Subcellular receptor forms and receptors from different porcine target organs react in the same fashion as do human, ovine, bovine, guinea pig, rabbit and rat estradiol receptors.

A human placental hormone (UTPH) with uterine growth and DNA promoting effects.

A human placental protein previously described is studied in order to expand its biological characterization. Dose-response-curve of its action on uterine growth of prepuberal mice showed to be a significant logarithmic function and this effect is neutralized by its specific antiserum. It is also demonstrated that the uterine growth-promoting effect is not due either to estrogen, protein-bound estrogen, progesterone or chorionic gonadotrophin. Experimental evidence is given to demonstrate that the mechanism of action of this placental protein is to stimulate the synthesis and to increase the concentration of DNA in uterine tissue, differing then in this respect from the effect of estrogen. Previous work has shown that this placental protein is present in full-term placentas within similar ranges as HCG and HPL. It is also detected in blood during pregnancy and acts biologically in at least two target organs: uterus and mammary gland. Therefore the name of uterotrophic placental protein (UTPH) has been suggested for this substance.