Competition for DNA steroid response elements as a possible mechanism for neuroendocrine integration

For the analysis of a simple steroid-dependent mating behavior, careful response definition, complete neural circuit delineation and placement of estrogen-responsive cells within this circuit have been accomplished. Molecular studies of two relevant genes have emphasized DNA/RNA hybridization assays nd DNA binding techniques. For both the rat preproenkephalin gene and the gene for the progesterone receptor, a strong induction by estrogen, tissue specificity of expression and a sex difference in regulation are prominent phenomena. On the rat preproenkephalin promoter, estrogen (ER) and thyroid receptors may compete for a DNA binding site. Likewise, progesterone (PR) and glucocorticoid receptors may compete for the same sites. On the rat PR gene, interactions between ER and AP-1 binding proteins are of special interest. Such interactions could underlay competitions and synergies between steroid hormones and neurally signalled events in the environment.     

Structure of eukaryotic DNA binding sites for steroid hormone-apolipoprotein A-I complexes

High affinity for DNA and synthetic oligonucleotides was detected for apolipoprotein A-I (ApoA-I) by affinity chromatography, affinity modification, and enzymatic analysis. Competitive inhibition and Southern hybridization showed that the tetrahydrocortisol (THC)-ApoA-I complex specifically bound to high-molecular-weight DNA in regions containing GCC/CGG sequences. The CC(GCC)₃ · GG(CGG)₃ duplex was found to be sensitive to nuclease S1 under the action of the THC-ApoA-I complex. The eukaryotic DNA binding sites for steroid (THC, androsterone)-ApoA-I complexes were found to be involved in the initiation of DNA copying in vitro.     

Ordered binding model as a general mechanistic mechanism for secondary active transport systems.

The mechanistic mechanism of secondary active transport processes has not been fully elucidated. Based on substrate binding studies dependent on coupling cation concentrations of the glutamate, melibiose, lactose and proline transport carriers in Escherichia coli, the ordered binding mechanism was proposed as the energy coupling mechanism of the transport systems. This ordered binding mechanism satisfactorily explained the properties of the secondary active transport systems. Thus, this mechanism as the general energy coupling mechanism for the transport systems is discussed.     

DNA as a possible target for antitumor ruthenium(III) complexes

The interaction of two experimental ruthenium(III)-containing antitumor complexes-Na[trans-RuCl(4)(DMSO)(Im)] (NAMI) and dichloro(1,2-propylendiaminetetraacetate)ruthenium(III) (RAP)-with DNA was investigated through a number of spectroscopic and molecular biology techniques, including spectrophotometry, circular dichroism, gel shift analysis, and restriction enzyme inhibition. It was found that both complexes slightly alter DNA conformation, modify its electrophoretic mobility, and inhibit DNA recognition and cleavage by some restriction enzymes, though they were less effective than cisplatin in producing such effects. Notably, the effects produced by NAMI on DNA were much larger than those induced by RAP. Implications of these results for the mechanism of action of ruthenium(III) antitumor complexes are discussed.     

Glucocorticoid receptor binding to calf thymus DNA. 1. Identification and characterization of a macromolecular factor involved in receptor-steroid complex binding to DNA.

Activation of receptor-steroid complexes to a form with high affinity for DNA is a poorly understood process involving multiple components in addition to the holoreceptor. Employing rat HTC cells as the source of glucocorticoid receptor, we show that maximal receptor binding to calf thymus DNA is mediated by a previously unknown small molecular weight factor. This factor can be removed from cytosolic preparations of receptor by gel filtration chromatography. Salt extraction of crude nuclear pellets afforded much larger amounts of a similar DNA-binding activity factor. The cytoplasmic factor and the more abundant nuclear factor were identical on the basis of their similar physical properties. The factor was precipitable in the crude state with (NH4)2SO4 and stable to heat as well as freezing and thawing. Chromatography on DNA-cellulose revealed that the factor itself did not bind to DNA. The factor could be filtered through a Centricon C-3 microconcentrator (molecular weight cutoff approximately 3000) but was excluded from Sephadex G-10 columns. These parameters enable us to determine an apparent molecular weight of 700-3000 for this factor. The presence of large amounts of this factor in nuclei accounts for the previously unexplained observation that, following size exclusion chromatography, more activated complexes bind to nuclei than to DNA. These data indicate that some, but not all, of the activated complexes require factor to be able to bind to DNA. The predominantly nuclear localization of this factor, coupled with its ability to increase DNA binding, attests to the biological relevance of this factor in the whole cell action of receptor-glucocorticoid complexes.     

Spin assay as a general method for studying plasma protein binding. Bilirubin-albumin binding.

A mutant of the Chinese Hamster Ovary cell line, CHO-K1, has been isolated which is resistant to killing by 25-hydroxy cholesterol in the absence of cholesterol. There is no effect on acetate incorporation into cholesterol by 25-hydroxy cholesterol in the mutant under conditions in which incorporation is inhibited in the parent cell. The mutant also appears to be defective in the regulation of cholesterol synthesis by serum choleserol.     

Intracellular receptors for activated protein kinase C. Identification of a binding site for the enzyme.

Protein kinase C (PKC) isozymes comprise a family of cytosolic enzymes that translocate to different intracellular sites on activation. We have recently characterized at least two intracellular receptor proteins for PKC (termed RACKs for receptors for activated C-kinase) in the Triton-insoluble material of the particulate fraction from neonatal rat heart. Here, we identify a sequence that appears to resemble the PKC binding site on these RACKs. A peptide (peptide I) with the sequence KGDYEKILVALCGGN bound PKC, and binding was markedly increased in the presence of PKC activators. Furthermore, peptide I inhibited PKC binding to RACKs in a dose-dependent manner. These data suggest that these RACKs have a common PKC binding sequence. Since peptide I inhibited PKC binding to RACKs in vitro, it may be a useful tool to inhibit PKC translocation and subsequent function in vivo.     

DNA structural dynamics: longitudinal breathing as a possible mechanism for the B in equilibrium Z transition.

The transition from B-DNA to Z-DNA not only requires a change in the helix architecture from a right-handed to a left-handed form, but also requires that all of the base pairs be flipped over. A transition mechanism is proposed in which the base pairs flip over one at a time while keeping the Watson-Crick hydrogen bonds intact. The two base pairs on either side of the pair that turns over form a kind of dynamic sandwich around it, and the flip takes place in a cavity that is formed when the distance between them is increased from about 7A in the intact helix to about 14A in the transition state. Since the transition occurs in two steps (formation of the cavity, followed by the base pair flip) and since the cavity propagates down the helix after the base pair is turned over, the mechanism accounts for the cooperativity of the B in equilibrium Z transition.     

DNA interpolyelectrolyte complexes as a tool for efficient cell transformation.

A tool was developed for enhancement of plasmid penetration into an intact cell, based on increasing DNA hydrophobicity via inclusion into a soluble interpolyelectrolyte complex (IPC) with polycations. The characteristics of formation of DNA IPC with synthetic polycations [poly(N-ethyl-4-vinylpyridinium)bromide (PVP) and PVP modified with 3% of N-cetyl-4-vinylpyridinium units (PVP-C)] were studied using ultracentrifugation and polyacrylamide gel electrophoresis methods. The conditions were established under which the mixing of DNA and polycation aqueous solutions results in the self-assembly of soluble IPC species. Incorporation of DNA into IPC results in the enhancement of DNA binding with isolated Bacillus subtilis membranes. A considerable increase in the efficiency of transformation of B. subtilis cells with pBC16 plasmid resulted from incorporation of the plasmid into the IPC with PVP and CVP.     

A new mechanism for steroid unresponsiveness: loss of nuclear binding activity of a steroid hormone receptor

The glucocorticoid, dexamethasone, binds to the specific cytosol receptors of a steroid-resistant mouse lymphoma cell line with the same affinity as to the receptors of the steroid-responsive parental cells. In the sensitive cells, the receptor-steroid complex translocates to the nucleus, whereas in the resistant cells nuclear transfer is greatly diminished. “Activated” receptor-dexamethasone complex from sensitive cells binds to isolated nuclei from both sensitive and resistant cell types, whereas the complex from the resistant cells binds to neither nuclei. Furthermore, the activated complex from sensitive cells binds to isolated homologous and heterologous DNA, whereas the complex from the resistant cells displays greatly reduced binding activity, implying that DNA plays a significant role in nuclear binding. These results suggest that the normal glucocorticoid receptor has two active domains: one for steroid binding, and the other for interaction with nuclear acceptor sites. The resistant cells described in this paper contain a receptor apparently defective in the latter activity.     

Phosphorylation of rhodopsin as a possible mechanism of adaptation.

Light-induced phosphorylation of rhodopsin has been extensively studied by a number of investigators from a biochemical point of view. However, little is known about the physiological function of this reaction. The slow rates measured for phosphorylation and dephosphorylation suggest that it may be involved in visual adaptation rather than in excitation. This paper presents biochemical data obtained from phosphorylation experiments in isolated photoreceptor membranes as well as in the physiological system of whole retinas and living animals. An attempt is made to compare the phosphorylation reaction with visual adaptation hypotheses taken from the electrophysiological literature. Finally, effects of cyclic nucleotide metabolism on the sensitivity of photoreceptors are presented and discussed.     

Functional heterogeneity in a multipinnate muscle.

Many mammalian muscles have a complex internal architecture. This type of structure could allow a single muscle to produce a variety of force vectors through selective regional contractions. This hypothesis was tested electromyographically in the multipinnate pig masseter by recording simultaneously from several intramuscular sites. It was found that the activity in different portions of the masseter varied systematically during the various phases of mastication. anatomical correlates of the differential activity included fasciculus orientation and length, sarcomere length in specific jaw positions, and histochemical fiber type. The usual assumptions made about muscles for biomechanical analysis, such as uniform contraction and constant line of action, are inappropriate for complex muscles such as the pig masseter.     

Mechanisms controlling steroid receptor binding to specific DNA sequences.

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.     

Functional heterogeneity among the T-derived lymphocytes of the mouse: III. Helper and suppressor T-cells activated by concanavalin A

We have studied the properties of T-cells which when activated by concanavalin A (Con A) either suppress or help the in vitro humoral response of mouse spleen cells. Previously established criteria for the T-cell populations, T₁ and T₂ were applied. T₁ cells were defined by their short half-life (2–3 wk) after adult thymectomy (ATx) and their resistance to small doses of antithymocyte serum (ATS). T₂ cells were defined by their long half-life (~15 wk) and their high sensitivity to ATS. T-cells which could be activated by Con A to help the response to the thymus-dependent antigen, sheep red blood cells, were found mainly in the T₂ subpopulation. T-cells which could be activated by Con A to suppress the response to the thymus-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), were found within both the T₁ and T₂ subpopulations.These results, our previous results, and those of others suggest that the T-cell responses to phytomitogens distinguish precursors committed to different functions, while the T₁ and T₂ classifications distinguish T-cells at different stages of maturation.     

Cutting of insertions from DNA molecules during heteroduplex formation as a possible mechanism for maintaining genome stability

A hypothesis has been put forward suggesting gene conversion as a mechanism of the cutting of foreign insertions (transposons, duplications) from DNA. During heteroduplex formation insertions result in one strand loops that can be attacked by nucleases specific for one strand DNA. A possible correlation is supposed to exist between the high activity of nucleases, on the one hand, and the low DNA content of genome and the absence of progressive evolution of the taxon, on the other hand.