Robot-assisted radical cystectomy with totally intracorporeal urinary diversion: surgical and early functional outcomes through the learning curve in a single high-volume center.

The aim of the study is to report surgical and early functional outcomes of first 100 patients undergoing robot-assisted radical cystectomy (RARC) with totally intracorporeal urinary diversion (ICUD) in a single center. The main surgeon (A.P.) attended a modular training program at a referring center mentored by a worldwide-recognized robotic surgeon (P.W.). The program consisted of: (a) 10 h of theoretical lessons; (b) video session (c) step-by-step in vivo modular training. Each procedure was performed as taught, without any technique variation. Demographics, intra-operative data and post-operative complications, along with early functional outcomes, were recorded for each patient. We retrospectively evaluated the first consecutive 100 patients submitted to RARC with totally ICUD from July 2015 to December 2018. Median age at surgery was 69 years (IQR 60-74). 52 (52%), 32 (32%), and 17 (17%) patients received orthotopic neobladder, ileal conduit and uretero-cutaneostomy, respectively. Median operative time was 410 min. A median number of lymph nodes retrieved were 27 and median estimated blood loss was 240 mL with median hospitalization time of 7 days. All procedures were completed successfully without open conversion. A statistically significant improvement was found in the late (30-90 post-operative days) post-operative complications (p = 0.02) and operative time for urinary derivation. At multivariate logistic regression model ASA score ≥ 3 (OR = 4.2, p = 0.002) and number of lymph nodes retrieved (OR = 1.16, p = 0.02) were found to be predictors of 90-day complications. An adequate modular training is paramount to obtain successful results and reduce the learning curve of RARC, as demonstrated by our experience.

Epigenetic control and the circadian clock: linking metabolism to neuronal responses.

Experimental and epidemiological evidence reveal the profound influence that industrialized modern society has imposed on human social habits and physiology during the past 50 years. This drastic change in life-style is thought to be one of the main causes of modern diseases including obesity, type 2 diabetes, mental illness such as depression, sleep disorders, and certain types of cancer. These disorders have been associated to disruption of the circadian clock, an intrinsic time-keeper molecular system present in virtually all cells and tissues. The circadian clock is a key element in homeostatic regulation by controlling a large array of genes implicated in cellular metabolism. Importantly, intimate links between epigenetic regulation and the circadian clock exist and are likely to prominently contribute to the plasticity of the response to the environment. In this review, we summarize some experimental and epidemiological evidence showing how environmental factors such as stress, drugs of abuse and changes in circadian habits, interact through different brain areas to modulate the endogenous clock. Furthermore we point out the pivotal role of the deacetylase silent mating-type information regulation 2 homolog 1 (SIRT1) as a molecular effector of the environment in shaping the circadian epigenetic landscape.

Nerve growth factor and its receptors TrkA and p75 are upregulated in the brain of mdx dystrophic mouse.

Increased angiogenesis and an altered blood-brain barrier have been reported in the brain of dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy. To further elucidate the mechanisms underlying angiogenesis in Duchenne muscular dystrophy, in this study we evaluated whether nerve growth factor (NGF) and nerve growth factor receptors (NGFRs) are involved, then correlated NGF-NGFRs expression with vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) content and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activity, by confocal laser microscopy and immunohistochemistry. Results showed that neurons, astrocytes and ependymal cells were strongly labeled by NGF in mdx brain, expressing NGFRs on glial and endothelial cells. In controls, NGF faintly labeled neurons and astrocytes, whereas endothelial cells were negative for NGFRs. Immunogold electron microscopy demonstrated NGFR gold particles on endothelial cells in mdx brain, while in controls few particles were recognizable only on glial end feet. Western blotting and real time polymerase chain reaction (RT-PCR) demonstrated a higher expression of NGF and NGFR mRNA and protein in mdx brain as compared to controls, and increase of VEGF-VEGFR-2 and active MMP-2 and -9 content. Overall, these data suggest that in the brain of mdx mice, an upregulation of the NGF-NGFRs system might be involved directly, or indirectly through the activation of VEGF-VEGFR-2 and MMP-2 and -9, in the angiogenic response taking place in this pathological condition.

Treatment of achalasia: lessons learned with Chagas' disease.

Chagas' disease (CD) is highly prevalent in South America. Brazilian surgeons and gastroenterologists gained valuable experience in the treatment of CD esophagopathy (chagasic achalasia) due to the high number of cases treated. The authors reviewed the lessons learned with the treatment of achalasia by different centers experienced in the treatment of Chagas' disease. Preoperative evaluation, endoscopic treatment (forceful dilatation and botulinum toxin injection), Heller's myotomy, esophagectomy, conservative techniques other than myotomy, and reoperations are discussed in the light of personal experiences and review of International and Brazilian literature. Aspects not frequently adopted by North American and European surgeons are emphasized. The review shows that nonadvanced achalasia is frequently treated by Heller's myotomy. Endoscopic treatment is reserved to limited cases. Treatment for end-stage achalasia is not unanimous. Esophagectomy was a popular treatment in advanced disease; however, the morbidity/mortality associated to the procedure made some authors seek different alternatives, such as Heller's myotomy and cardioplasties. Minimally invasive approach to esophageal resection may change this concept, although few centers perform the procedure routinely.

Regulation of human MC2-R gene expression by CREB, CREM, and ICER in the adrenocortical cell line Y1.

The MC2-Receptor (melanocortin 2 receptor, MC2-R) is a Gs-protein coupled receptor that is upregulated by its own ligand ACTH and by forskolin. The mechanisms regulating MC2-R expression are still unclear. We therefore investigated the role of the stimulatory transcription factors CREB and CREM and the inhibitory factor ICER for regulation of human MC2-R expression. We cotransfected mouse adrenocortical Y1 cells with luciferase reporter gene vectors containing full length and deleted human MC2-R promoter constructs with expression plasmids for CREB, CREBS133A, CREMtau, CREMtauS117A, or ICER. Direct protein-DNA interaction was investigated by EMSA. Wild type CREB did not significantly affect promoter activity due to high endogenous CREB activity. However, CREBS133A decreased forskolin stimulated MC2-R promoter activity by 48+/-5% (mean+/-SEM) while unstimulated values remained unchanged. CREMtau moderately increased basal and forskolin stimulated luciferase activity in a dose-dependent manner (maximum effect 252+/-24% and 186+/-13% VS. control vector, respectively). While this effect required the full length promoter, cAMP stimulation was retained in shorter constructs. ICER reduced basal luciferase activity in Y1 cells by 17+/-28%, but completely abolished forskolin stimulation. Although 5'-deletion constructs mapped the minimum promoter region required for ICER effect to the shortest -64/+40 construct, direct protein DNA interaction in this promoter region could not be identified by EMSA. Moreover, mutation of the SF-1 binding sites, which retained ICER dependent inhibition, excluded SF-1 to be required for this effect. We conclude from these data that transcription factors of the CREB/CREM/ATF family have a moderate effect on human MC2-R promoter activity, but seem to play a minor role in transmitting stimulation of the cAMP pathway to increased MC2-R expression.

Increased matrix-metalloproteinase-2 and matrix-metalloproteinase-9 expression in the brain of dystrophic mdx mouse.

Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.

Combinations of genetic changes in the human cAMP-responsive element modulator gene: a clue towards understanding some forms of male infertility?

The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon gamma). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon gamma. Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility.

Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1.

Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.

[Promoter effect of mercury chloride and methyl-mercury on human keratinocytes in culture]. / Effetto promoter del mercurio cloruro e del metil-mercurio su cheratinociti umani in coltura.

Mercury has received considerable media focus because it is present in dental amalgams and seafood. There is potential exposure in gas meters, thermometers and fluorescent lamps workers. To evaluate its possible epigenetic carcinogen effect, cultures of human keratinocytes were treated with increasing doses of HgCl2 for 30 min, 24 h and of CH3HgCl for 24 h, respectively. The red neutral method was used to evaluate the doses of HgCl2 and CH3HgCl which had no cytotoxic effect. Then, the dye transfer method was used to investigate the gap junctions-mediated intercellular communication (GJIC). Cells were microinjected with Lucifer Yellow CH by using the Eppendorf Apparatus and the Leica inverted microscope. After 30 min incubation at the concentration of 10 microM, HgCl2 did not exert inhibition of GJIC. Conversely, after 24 h at the concentration of 10 nM, HgCl2 inhibited GJIC. Incubation with CH3HgCl at the concentration of 250 nM for 24 h reduced the number of fluorescent cells, thus denoting a inhibition of GJIC. Taken together our data demonstrated that: i) HgCl2 and CH3HgCl exerted an inhibitory effect upon GJIC; ii) HgCl2 resulted to inhibit GJIC at concentrations 25 folds lower than CH3HgCl. Further studies will be addressed to evaluate whether the reversal of GJIC inhibition could be obtained by withdrawal of toxic substance, or by the addition of a GJIC activator like the retinoic acid. Finally to shed light on the possible effect of mercury derivates at the transcriptional or translational levels, the expression profile of the connexin 43 gene after HgCl2 and CH3HgCl exposure of cultured human keratinocytes will be investigated.

Altered behavioral rhythms and clock gene expression in mice with a targeted mutation in the Period1 gene.

A group of specialized genes has been defined to govern the molecular mechanisms controlling the circadian clock in mammals. Their expression and the interactions among their products dictate circadian rhythmicity. Three genes homologous to Drosophila period exist in the mouse and are thought to be major players in the biological clock. Here we present the generation of mice in which the founding member of the family, Per1, has been inactivated by homologous recombination. These mice present rhythmicity in locomotor activity, but with a period almost 1 h shorter than wild-type littermates. Moreover, the expression of clock genes in peripheral tissues appears to be delayed in Per1 mutant animals. Importantly, light-induced phase shifting appears conserved. The oscillatory expression of clock genes and the induction of immediate-early genes in response to light in the master clock structure, the suprachiasmatic nucleus, are unaffected. Altogether, these data demonstrate that Per1 plays a distinct role within the Per family, as it may be involved predominantly in peripheral clocks and/or in the output pathways of the circadian clock.

Heat shock interferes with steroidogenesis by reducing transcription of the steroidogenic acute regulatory protein gene.

A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.

The closely related POU family transcription factors Brn-3a and Brn-3b are expressed in distinct cell types in the testis.

Although the Brn-3a and Brn-3b POU family transcription factors were originally identified in neuronal cells, their expression in some non neuronal cell types has previously been reported. Here we report that Brn-3a and Brn-3b are also expressed in the testis with expression of each factor being observed at distinct stages of germ cell development. Thus, Brn-3a is expressed in spermatogonia whereas Brn-3b expression is observed in post-meiotic spermatids. In agreement with this, Brn-3a expression is detectable much earlier than that of Brn-3b in testes derived from sexually immature postnatal animals. Similarly, Brn-3b expression is absent in knock out mice lacking a functional CREM transcription factor in which the later stages of germ cell development do not occur, whereas Brn-3a expression is observed at similar levels in the testes of these knock out mice. Interestingly, the cellular pattern of Brn-3a expression during germ cell development coincides with that of the BRCA-1 anti-oncogene. Consistent with the possibility that Brn-3a may regulate expression of BRCA-1 in the testis, we have shown that Brn-3a can strongly activate the BRCA-1 promoter in co-transfection experiments whereas Brn-3b does not have this effect. Hence, as observed in neuronal cells, Brn-3a and Brn-3b may play distinct and important functional roles in the regulation of gene expression during germ cell development.

Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways.

Corticotropin-releasing factor (CRF), a neuropeptide of 41 amino acids, acts as the major physiological regulator of the basal and stress-induced release of corticotropin (ACTH), beta-endorphin and other proopiomelanocortin-derived peptides from the anterior pituitary gland. In addition to its endocrine activity, CRF displays extrahypophysiotropic effects, mainly as a regulator of stress responses. We show here that CRF may additionally function as a differentiating factor in immortalized noradrenergic neuronal CATH.a cells that express CRF receptor type I and resemble locus coeruleus-derived neurons. CRF triggers morphological changes in CATH.a cells including the appearance of extended long, slender neurites with prominent growth cones. CRF-treated CATH.a cells exhibit a morphology similar to locus coeruleus neurons in primary culture. CRF-induced neurite outgrowth of CATH.a cells was blocked by addition of inhibitors for cAMP-dependent protein kinase or extracellular signal-regulated protein kinase (ERK), a subtype of the mitogen-activated protein kinases. The participation of ERK within the CRF signalling cascade was further confirmed by Western blot experiments, with antibodies directed against the phosphorylated form of ERK, and also with transcription-based assays. We conclude that CRF functions as a differentiating factor of CATH.a cells via the cAMP and the MAP kinase signalling pathways.

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells.

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.

Light induces chromatin modification in cells of the mammalian circadian clock.

The mammalian circadian clock resides in neurons of the hypothalamic suprachiasmatic nucleus (SCN). Light entrains phase resetting of the clock using the retino-hypothalamic tract, via release of glutamate. Nighttime light exposure causes rapid, transient induction of clock and immediate-early genes implicated in phase-shifting the pacemaker. Here we show that a nighttime light pulse caused phosphorylation of Ser10 in histone H3's tail, in SCN clock cells. The effect of light was specific, and the kinetics of H3 phosphorylation were characteristic of the early response, paralleling c-fos and Per1 induction. Using fos-lacZ transgenic mice, we found that H3 phosphorylation and Fos induction occurRed in the same SCN neurons. Systemic treatment with the GABAB receptor agonist baclofen prevented light-induced c-fos and Per1 expression and H3 phosphorylation, indicating that one signaling pathway governs both events. Our results suggest that dynamic chromatin remodeling in the SCN occurs in response to a physiological stimulus in vivo.

Activation of RSK by UV-light: phosphorylation dynamics and involvement of the MAPK pathway.

Ribosomal S6 kinases (RSKs) are serine/threonine kinases activated by mitogenic signals through the Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinases (MAPK/ERK). RSKs contain two heterologous complete protein kinase domains. Phosphorylation by ERK of the C-terminal kinase domain allows activation of the N-terminal kinase domain, which mediates substrate phosphorylation. In human, there are three isoforms of RSK (RSK1, RSK2, RSK3), whose functional specificity remains undefined. Importantly, we have shown that mutations in the RSK2 gene lead to the Coffin-Lowry syndrome (CLS). In this study, we characterize two monoclonal antibodies raised against phosphorylated forms of the N- and C-terminal domain of RSK2 (P-S227 and P-T577, respectively). Using these two antibodies, we show that stress signals, such as UV light, induce phosphorylation and activation of the three RSKs to an extent which is comparable to Epidermal Growth Factor (EGF)-mediated activation. The use of specific kinase inhibitors indicates that UV-induced phosphorylation and activation of RSK2 is mediated by the MAPK/ERK pathway, but that the Stress-Activated Protein Kinase 2 (SAPK2)/p38 pathway is also involved. These results modify the view of RSKs as kinases restricted to the mitogenic response and reveal a previously unappreciated role of MAPKs in stress induced signaling. Oncogene (2000) 19, 4221 - 4229

Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that Is independent of cholesterol side-chain cleavage.

An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.

Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation.

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. Here we report that these two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these dimodified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression.