Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis.

The modulation of glucocorticoid receptor activity by cyclic nucleotides was studied in cultured human skin fibroblasts. The receptors appeared to be activated in the presence of dibutyryl-cAMP and inactivated by dibutyryl-cGMP. Significantly, the cGMP content of the fibroblasts increased during cell growth, with a concomitant decrease in the glucocorticoid receptor activity, while when the cells reached early confluency the decrease in cGMP content was accompanied by an increase in cAMP and increased activity of the glucocorticoid receptors. In addition, cortisol induced (2'-5')oligoadenylate synthetase in these cells and raised the cellular (2'-5')oligoadenylate concentrations. This resulted in a decrease in both DNA and protein synthesis activity in the cells, a response which correlated with the (2'-5')oligoadenylate concentration. The combination of cortisol and dibutyryl-cAMP had a synergetic stimulatory effect on the (2'-5')oligoadenylate concentration and a synergetic inhibitory effect on protein synthesis. In conclusion, it is demonstrated here that cyclic nucleotides can modulate glucocorticoid receptor activity in cultured human skin fibroblasts, and thus these compounds may indirectly affect cellular metabolism by regulating the cellular responses to glucocorticoids.

Specific non-enzymatic glycation of the rat histone H1 nucleotide binding site in vitro in the presence of AlF4-. A putative mechanism for impaired chromatin function.

We show here that an aluminium derivative, AlF4-, stimulates glycation of histone H1 selectively in the proximity of its nucleotide-binding site. This adduct formation interferes with nucleoside triphosphate hydrolysis by H1 and with nucleotide modulation of H1 DNA binding. The present mode of aluminium action may in part be responsible for its effects on the chromatin structure and expression of tissue-specific genes, and may constitute a mechanism in the pathogenesis of aluminium-induced encephalopathy and in that of Alzheimer's disease, for example.

Cortisol decreases the cellular concentration of translatable procollagen mRNA species in cultured human skin fibroblasts.

The effect of cortisol on the cellular concentration of translatable procollagen mRNAs was studied in cultured human skin fibroblasts. Cortisol selectively decreased the amount of procollagen mRNAs, in comparison to the total mRNA activity, when the cells were grown in enriched medium conditions, i.e., with 10% newborn calf serum. The selective decrease was first observed after 6 h exposure to 1 microM cortisol. In depleted medium conditions, i.e., with 2% newborn calf serum, the initial response was a stimulatory one, followed after about 12 h by a decrease in the procollagen mRNA activity. The results suggest that the selective inhibitory effect of cortisol on the cellular concentration of translatable procollagen mRNA species needs an optimal serum concentration. Furthermore, the results give support to the hypothesis that the decrease in the procollagen mRNA concentration after cortisol administration is a secondary response, preceded by the induction of some intracellular regulation system.

Coordination of transcription of the human 17 beta-hydroxysteroid dehydrogenase type 1 gene (EDH17B2) by a cell-specific enhancer and a silencer: identification of a retinoic acid response element.

Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes primarily the reductive reaction of estrone to the biologically more active form, estradiol. The enzyme is highly expressed in the human placenta and the ovary and, in addition, in certain estrogen target cells, such as breast epithelial cells. To elucidate the transcriptional control of the EDH17B2 gene, the gene encoding 17HSD type 1, we fused a series of 5'-deletion mutants of the EDH17B2 gene into chloramphenicol acetyl transferase reporter gene vectors. An enhancer region was identified within the bases -661 to -392 and it increased, in both orientations, thymidine kinase promoter activity more than 200-fold in JEG-3 choriocarcinoma cells. This enhancer region was also functional in another choriocarcinoma cell line, JAR, although to a lesser extent. In BT-20 and T-47D breast cancer cells the enhancer region increased thymidine kinase promoter activity to some degree but not as efficiently as expected on the basis of endogenous enzyme expression. No such enhancer activity was observed in 17HSD type 1 nonexpressing cell lines. The retinoic acid responsive element, which was located between bases -503 and -487 in the EDH17B2 enhancer, bound retinoid acid receptor alpha retinoid X receptor alpha complex and transmitted retinoic acid induction on transcription in JEG-3 and T-47D cells. Finally, a silencer, functional in all the cell lines tested, was localized in the region from -392 to -78. Deletion of the region lad to a 4-fold increase in reporter gene expression. Altogether, our findings suggest that transcriptional control of the EDH17B2 gene is coordinated by the cell-specific enhancer and the silencer.

Steroid-involved transcriptional regulation of human genes encoding prostatic acid phosphatase, prostate-specific antigen, and prostate-specific glandular kallikrein.

We have compared the steroid regulation of human genes encoding prostatic acid phosphatase (hPAP), prostate-specific antigen (hPSA), and prostate-specific glandular kallikrein (hK2) at the level of transcription. Reporter constructs of hPAP promoter covering the region -734/+467 were functional in both prostatic (LNCaP and PC-3) and nonprostatic (CV-1) cell lines in transient transfections. hPAP -231/+50 with eight identified transcription factor-binding sites showed the highest, and hPAP -734/+467 showed the lowest transcriptional activity in CV-1 cells. The hPAP promoter could not be induced with androgen, glucocorticoid, or progesterone, contrary to the hPSA (-620/+40) and hK2 (-493/+27) promoters in PC-3 cells cotransfected with the respective steroid receptor expression vector. Therefore, steroids cannot directly regulate hPAP gene expression via receptor binding to steroid response elements at -178 and +336, which have been shown to have androgen receptor-binding ability in vitro. Glucocorticoid was the most powerful activator of the hPSA construct at 10-nM steroid concentrations. On the contrary, glucocorticoid stimulation of the transcriptional activity of the hK2 construct was the weakest among the tested steroids. The results indicate that the steroid response elements in the proximal promoters of hPSA and hK2 genes are not androgen specific, offering the molecular basis for the expression of these genes outside the prostate in tissues containing steroid receptors.

Histone H1 and the regulation of transcription by nuclear receptors.

Histone H1 is a eukaryotic repressor which recognizes specific DNA structures, and nucleotides regulate its interaction with DNA. Since their mode of action may be considered similar to that observed in the case of plasma membrane GTPases, H1 may be regarded as an ATP/GTPase involved in the action of nuclear receptors. A hypothesis is put forward here to suggest that transcriptional activators CTF/NF-I and AP-1 (fos/jun), for example, are effectors for H1. H1 and CTF/NF-I may be members of a stimulatory regulatory cascade for nuclear receptor action that ends with selective activation of chromatin through histone modification and the disruption or a more subtle structural change of a specific nucleosome, while an opposite effect may be obtained through modification of fos/jun by H1.

The histone H1-lacZ' fusion protein produced in Escherichia coli binds to the 5'-TTGGCAnnnTGCCAA-3' motif on DNA.

The coding region of the chicken histone H1.03 gene was cloned to a bacterial expression vector, and the 291-amino acid H1-beta-galactosidase fusion protein was isolated after induction with IPTG. The fusion protein recognizes the 5'-TTGGCAnnnTGCCAA-3' motif on DNA. The H1 globular domain was initially shown to be responsible for the sequence-specific binding by functional deletion analysis. This function may be indispensable for the role of H1 as a determinant of nucleosome positioning and as a eukaryotic repressor.

Nucleotide and calcium-induced conformational changes in histone H1.

The principal constituents of chromatin, histone H1 (H1) and the nucleosome have essential roles in regulation of eukaryotic gene expression. However, mechanisms for the H1-dependent inactivation and for the ATP-dependent chromatin remodeling upon activation are largely unelucidated. Using circular dichroism (CD) analysis we show that ATP and other nucleotides and Ca2+ induce structural changes in H1. ATP and Ca2+ also induce changes when H1 is interacting with DNA, and the changes in H1 are accompanied by alterations in its DNA interaction. These results suggest that nucleotide and Ca2+ binding may be important for H1-mediated chromatin changes.

The predicted molecular structure suggests that CTF/NF-I may function as a histone acetylase.

The primary structure of nuclear factor-I (CTF/NF-I), a eukaryotic regulatory DNA-binding protein involved in both DNA replication and gene transcription, and the secondary structure predictable from it, are compared here with those of a number of prokaryotic acetylases. Hydropathy and Chou-Fasman analyses reveal that the polypeptide chain of CTF/NF-I is likely to fold to higher order structures similar to those of the acetylases, and significant conservation of functionally important regions of the acetylases is observed in CTF/NF-I. It is therefore suggested that CTF/NF-I may function as a histone acetylase.

DNA binding of histone H1 is modulated by nucleotides.

Histone H1 acts as a general repressor of transcription in eukaryotes by organizing nucleosomes into inaccessible condensed forms of chromatin. The capability of H1 to bind to DNA with some sequence specificity is likely to be critical in the control of these processes. We show here that ATP and several other nucleotides, including non-hydrolyzable derivatives, can inhibit DNA binding of H1. The results also show that ATP differentially affects binding of H1 to DNA in a fashion enhancing nucleotide sequence specificity of the binding. The study suggests a novel mechanism of modulation of H1 activity that has important implications for the role of H1 as a transcriptional regulator.

Interference of AlF4- with nucleotide and DNA binding of rat histone H1 in vitro. Implications for the pathogenesis of Alzheimer's disease.

We demonstrate here that the H2PO4- analogue AlF4- binds to the nucleotide-binding site of rat liver histone H1 in vitro, and interferes with nucleotide recognition and H1 DNA binding. AlF4- may thus compromise the genetically determined pattern of protein synthesis through binding to H1, the general repressor. The present findings are of interest as a number of studies have implicated aluminium as a factor in the pathogenesis of Alzheimer's disease.

Ultrasound-controlled neuronavigator-guided brain surgery.

The development of a unique neurosurgical navigator is described and a preliminary series of seven cases of intracerebral lesions approached with the assistance of this neuronavigation system under ultrasound control is presented. The clinical series included five low-grade astrocytomas, one chronic intracerebral hematoma, and one porencephalic cyst. Management procedures included biopsy in all cases, drainage of the hematoma, and endoscopy and fenestration for the cyst. The features of the neuronavigation system are interactive reconstructions of preoperative computerized tomography and magnetic resonance imaging data, corresponding intraoperative ultrasound images, versatility of the interchangeable end-effector instruments, graphic presentation of instruments on the reconstructed images, and voice control of the system. The principle of a common axis in the reconstructed images served to align the navigational pointer, biopsy guide, endoscope guide, ultrasound transducer, and surgical microscope to the brain anatomy. Intraoperative ultrasound imaging helped to verify the accuracy of the neuronavigator and check the results of the procedures. The arm of the neuronavigation system served as a holder for instruments, such as the biopsy guide, endoscope guide, and ultrasound transducer, in addition to functioning as a navigational pointer. Also, the surgical microscope was aligned with the neuronavigator for inspection and biopsy of the hematoma capsule to rule out tumor etiology. Voice control freed the neurosurgeon from manual exercises during start-up and calibration of the system.

Impairment of histone H1 DNA binding by adduct formation with acetaldehyde.

Incubation of histone H1 with pharmacologically relevant concentrations of acetaldehyde resulted in the formation of spontaneously stable acetaldehyde-protein linkages. The reaction of acetaldehyde and H1 purified from rat liver either by a DNA recognition site affinity chromatography or by perchloric acid extraction occurred primarily at the lysine residues in the carboxyterminal tail of H1, which is crucial for its function as a eukaryotic repressor. It was further shown using an H1-lacZ fusion protein produced in E. coli and the protein isolated from rat liver that the formation of acetaldehyde adducts with H1 impair its DNA binding properties. We propose that such a reaction may occur in vivo and lead to an inability to repress genes in the liver upon excessive alcohol consumption. This mechanism may play a role in acetaldehyde-induced collagen synthesis in alcoholics.

Interactions of Hsp90 with histones and related peptides.

The 90 kDa heat shock protein (Hsp90) induces the condensation of the chromatin structure [Csermely, P., Kajtár, J., Hollósi, M., Oikarinen, J., and Somogyi, J. (1994) Biochem. Biophys. Res. Commun. 202, 1657-1663]. In our present studies we used surface plasmon resonance measurements to demonstrate that Hsp90 binds histones H1, H2A, H2B, H3 and H4 with high affinity having dissociation constants in the submicromolar range. Strong binding of the C-terminal peptide of histone H1 containing the SPKK-motif and a pentaeicosa-peptide including the Hsp90 bipartite nuclear localization signal sequence was also observed. However, a lysine/arginine-rich peptide of casein, and the lysine-rich platelet factor 4 did not display a significant interaction with Hsp90. Histones and positively charged peptides modulated the Hsp90-associated kinase activity. Interactions between Hsp90, histones, and high mobility group (HMG) protein-derived peptides raise the possibility of the involvement of Hsp90 in chromatin reorganization during steroid action, mitosis, or after cellular stress.

A rapid and sensitive assay for protein disulphide isomerase activity.

An assay procedure for the determination of protein disulphide isomerase activity is presented. The method is based on the reactivation of randomly cross-linked RNAase, the extent of RNAase reactivation being determined from the degradation of radioactively labelled RNA. The method is rapid and sensitive and allows one to test a large number of samples simultaneously.

Volume rendering using seed-filling acceleration: supporting cut planes by fast reseeding.

OBJECTIVE: Seed filling in view lattice is a method for accelerating volume rendering. Our previously published results on using seed filling had the limitation that they did not support cut planes. Cut planes are one of the main advantages of using volume rendering over surface rendering. In this article we describe the seed-filling acceleration technique and propose two fast reseeding processes that may be performed for each frame. MATERIALS AND METHODS: The reseeding process performs a connected component search on the cut plane within the volume data set. The amount of processing done by a connected component search algorithm is proportional to N(2), assuming the data set size is N(3). We implemented two connected component search algorithms: one for orthogonal cut planes and another for oblique cut planes. We measured the time taken by the cutting and estimated the effect on the rendering performance when the cut plane was moved interactively. RESULTS: Our measurements show that rendering rates of several frames per second can be achieved with a 266-MHz Pentium II PC, even when the object is interactively modified with cut planes during the rendering. CONCLUSIONS: We have shown that the seed-filling algorithm is also applicable in situations where the displayed data is modified interactively using cut planes or objects. Applications in this regard include, for example, computer-aided surgery systems, where the instrument may be used to interactively perform the simulated cut in the data set.

Homologous structures of nuclear and GTPase-linked plasma membrane receptors suggest analogous mechanisms of action.

The molecular structures of nuclear and GTPase-linked plasma membrane receptors are compared here in the light of a recent finding suggesting that histone H1 may be an ATP/GTPase involved in transduction of the action of nuclear receptors. Considerable homology and conservation of the regions responsible for the interaction of the plasma membrane receptors with GTPases was observed in the nuclear receptors, thus suggesting analogous mechanisms of action and a common evolutionary origin for the two receptor families.

Experimental spinal fusion with decalcified bone matrix and deep-frozen allogeneic bone in rabbits.

The effects of allogeneic decalcified bone matrix (DBM) on the formation of bone between the spinous processes of the rabbit vertebrae was compared with the effects of allogenic deep-frozen cortical bone (AFB). Autologous cancellous bone (ACB) chips were used for a control substance. Healing was estimated by gross anatomical roentgenologic and microscopic methods one, two, four and six months after the operation. The ACB and DBM transplants gave comparable results. A stable bony bridge was formed at one month, and this was seen to strengthen during the follow-up time. Both transplanting materials were accepted by the host without foreign body reactions. Inflammatory cell accumulations and sequesterated particles were not seen in any of the specimens with ACB and DBM transplants. The DBM induced new bone formation and the transplanted material was resorbed within two months after the operation. The AFB transplants did not induce the new bone formation, but the implanted fragments in contact with the host bone were surrounded by the callus, which produced a bony bridge but only as late as four months after the operation. After six months, the bridging was incomplete and sequestered bone particles were still seen in some of the specimens. The AFB transplants were slowly resorbed and accumulations of inflammatory cells were present at least six months postoperation. The results indicate that in rabbits, decalcified bone matrix is a better substitute for autologous bone than the allogeneic deep-frozen cortical bone.