Pediatric patients with Marfan syndrome: frequency of dural ectasia and its correlation with common cardiovascular manifestations.

PURPOSE: Marfan syndrome (MFS) is a genetic disorder of the connective tissue. Aortic root dilation is a main criterion of the Ghent Nosology. Dural ectasia and the presence of mitral valve prolapse (MVP) contribute to its systemic score. The purpose of this study was to investigate the frequency of dural ectasia and its correlation with cardiovascular manifestations in a pediatric study population. PATIENTS AND METHODS: 119 pediatric patients with confirmed or suspected MFS were examined in the local Marfan Clinic. 31 children with MFS who underwent magnetic resonance imaging (MRI) were included. Each patient was evaluated according to the Ghent nosology. Echocardiography was used to measure the aortic root diameter and assess the presence of MVP and mitral regurgitation. Z-scores were calculated for the evaluation of the aortic root diameters. MRI was performed to determine the dural sac ratio (DSR). RESULTS: The prevalence of dural ectasia was 90.3 %, of aortic root dilation 32.2 %, of MVP 64.5 % and of mitral regurgitation 51.6 %. DSR at L5 correlated with the intraindividual z-scores (slope, 3.62 ±â€Š1.5 [0.56; 6.68]; r = 0.17; p = 0.02; F = 5.84). Z-scores ≥ 2 were accompanied by dural ectasia in 100 %, MVP in 95 % and mitral regurgitation in 100 % of cases. MVP was accompanied by mitral regurgitation in 70 % of cases. CONCLUSION: As the examined cardiac manifestations show a coincidence with dural ectasia in 95 - 100 % of cases, MRI for diagnostic dural sac imaging should be reserved for MFS suspicions with the absence of those manifestations in order to establish the diagnosis according to the Ghent criteria. Thus, the present study supports the recent downgrading of dural ectasia to a contributor to the systemic score.

Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors.

n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrate-induced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence of cell death to nearly 100% of the population, whereas HCT 116 cells largely remain alive during treatment with this combination. The action of butyrate as an inhibitor of histone deacetylase was assessed in these cell lines by examining extracted core histones for their electrophoretic mobility in Triton/acid/urea gels. The concentrations of butyrate that were effective for inducing apoptosis were similar to the concentrations that caused hyperacetylation of core histones in the VACO 5 cell line. Furthermore, an examination of other carboxylic acids for induction of apoptosis revealed a rank order that corresponded to the order of potency in causing hyperacetylation of core histones. Specifically, the active acids were 3-5 carbons in length and lacked substitution at the 2-position. Isovaleric and propionic acids, in particular, proved to be effective inducers of both hyperacetylation and apoptosis at 5 mM concentrations, a finding of potential relevance to the unusual pancytopenia occurring after acidotic episodes in isovaleric and propionic acidemias. The duration of butyrate treatment required for chromatin fragmentation (10-20 hr) corresponded to the time required for histone H4 to become predominantly tetraacetylated. Furthermore, trichostatin A, a structurally dissimilar inhibitor of histone deacetylase, mimicked butyrate-induced apoptosis of VACO 5 cells and growth inhibition of HCT 116 cells. The dramatic enhancement of VACO 5 cell death by TPA, and the high level resistance of HCT 116 cells to butyrate were not evident from histone acetylation determinations. Thus, applications of butyrate for cytoreduction therapy will benefit from pharmacodynamic assessment of histone acetylation, but will require additional work to predict susceptibility to butyrate-induced death.

Phorbol ester augments butyrate-induced apoptosis of colon cancer cells.

Butyrate is a potentially selective therapeutic agent for many adenocarcinomas. Butyrate causes reversible growth arrest as well as some death of VACO 5 colon cancer cells. Combined treatment with butyrate and the phorbol ester TPA leads instead only to cell death, while TPA causes little death on its own. Cells dying during treatment with TPA and butyrate, as well as those dying in the presence of butyrate alone, exhibit features typical of apoptosis, including detachment, shrinkage and internucleosomal DNA cleavage. Pre-treating VACO 5 cell cultures with TPA for as little as 6 hr prior to butyrate addition led to a markedly diminished enhancement of butyrate-induced apoptosis. Treatment with a distinct PKC activator, bryostatin 1, was ineffective in enhancing butyrate-induced death and, furthermore, counteracted the death-enhancing actions of TPA. Such antagonism was apparent when bryostatin was added after 12 hr of TPA/butyrate treatment but was much less effective thereafter. The duration of TPA/butyrate treatment required for depressing cell survival by >95% was thereby estimated to be 24 hr. Other colon cancer cell lines were examined for the extent of cell death following treatment with TPA/butyrate. In each of these lines, butyrate inhibited cell replication in a reversible manner, similar to that seen in VACO 5. However, the combination of butyrate and TPA led to high levels of cell death in only a subset of these lines. TPA/butyrate-treated cultures of COLO 201 exhibited extensive apoptosis, similar in timing and magnitude to the response by VACO 5, whereas HCT 116 was reversibly growth-arrested. Our findings indicate that the PKC system plays a critical role in maintaining cell survival during butyrate-induced growth arrest.

Estrogen receptor-dependent formation of two distinct multiprotein complexes on the human pS2 gene regulatory segment. Participation of a c-fos related protein.

DNA-protein interactions around the regulatory region of the pS2 gene were studied to gain insight into the mechanisms that operate in the estrogen receptor regulated expression of this gene in the MCF-7 human breast cancer cell. Using a revised photocrosslinking technology in combination with gel retardation assays, two distinct multiprotein DNA complexes were shown to assemble in an estrogen receptor-dependent process. Immunological analysis demonstrated the participation of both the estrogen receptor and a c-fos related protein in the formation of these complexes. The results support a model of estrogen receptor function in which the receptor facilitates the formation of multiprotein complexes at DNA sites that can regulate the transcription of a hormone responsive gene by RNA polymerase II and any additional general transcription machinery. These receptor-containing complexes are referred to as "receptorsomes."

Release of cell surface proteoglycans from differentiating colon cells proceeds by cleavage of lipophilic anchor peptides.

Heparan sulphate proteoglycans are rapidly released from VACO 10MS colon cancer cells that are triggered with phorbol esters to undergo terminal differentiation. This lag-free temperature-sensitive process is correlated with a conversion of the lipophilic proteoglycans of the cell surface into non-lipophilic proteoglycans that accumulate in the culture medium. The released proteoglycans are very similar to their lipophilic precursors in size, buoyant density and glycosaminoglycan characteristics; however, they exhibit slightly smaller core proteins after chemical and enzymic deglycosylation. The lipophilicity of the larger-sized core proteins of the cell-associated proteoglycans is also correlated with the presence of an easily iodinatable domain; this domain is missing in the released proteoglycans. Exogenous proteases (i.e. chymotrypsin, V8, trypsin and proteinase K) readily cleave this segment from the larger protease-resistant region of the proteoglycan structure. It is also released intact by treatment of the isolated proteoglycans with methanolic HCl. This component appears to be peptide in character, in that proteases readily degrade it and release iodotyrosines when the precursor has been iodinated. No evidence for the presence of covalently attached fatty acids in the cell-associated proteoglycans was found. These results are consistent with the hypothesis that the altered proteoglycan metabolism that is associated with the phorbol-ester-induced terminal differentiation of certain human colon cancer cells ensues upon the activation of a membrane-localized protease that cleaves a lipophilic anchor segment from the cell surface proteoglycans.

A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain.

Upon binding estrogen, the estrogen receptor (ER) is proposed to undergo some form of conformational transition leading to increased transcription from estrogen-responsive genes. In vitro methods used to study the transition often do not separate heat-induced effects on the ER from estrogen-induced effects. The technique of affinity partitioning with PEG-palmitate was used to study the change in the hydrophobic surface properties of the ER upon binding ligand with and without in vitro heating. Upon binding estradiol (E2), the full-length rat uterine cytosolic ER undergoes a dramatic decrease in surface hydrophobicity. The binding of the anti-estrogen 4-hydroxytamoxifen (4-OHT) results in a similar decrease in surface hydrophobicity. These effects are independent of any conformational changes induced by heating the ER to 30 degrees C for 45 min. The use of the human ER steroid binding domain overproduced in Escherichia coli (ER-C) and the trypsin-generated steroid binding domain from rat uterine cytosolic ER demonstrates that the decrease in surface hydrophobicity upon binding E2 or 4-OHT is localized to the steroid binding domain. Gel filtration analysis indicates that the change in surface hydrophobicity upon binding ligand is an inherent property of the steroid binding domain and not due to a ligand-induced change in the oligomeric state of the receptor. The decrease in surface hydrophobicity of the steroid binding domain of the ER upon binding E2 or 4-OHT represents an early and possibly a necessary event in estrogen action and may be important for "tight" binding of the ER in the nucleus.

Overproduction of full-length and truncated human estrogen receptors in Escherichia coli.

The full-length human estrogen receptor (hER) as well as two overlapping peptides of hER were overproduced in Escherichia coli JM109 cells, using the inducible pIC vector system. The N-terminal receptor peptide contains the DNA-binding domain as well as the hinge region, whereas the C-terminal peptide contains the same hinge region and the hormone-binding domain. Typically, 1-6 mg of estrogen receptor (ER) peptides can be recovered from 1 L E. coli cell cultures. The majority of the overexpressed proteins are found in inclusion bodies, which allow the isolation of ER peptides in high yields and of 50-80% purity. Induction for short time periods at 10 microM inducer yielded up to 50% of the ER peptides in soluble form with full biological activity. Both the intact receptor and the C-terminal fragment specifically bound estrogens and antiestrogens, whereas ER peptides that contained the DNA-binding domain were retained on a DNA-agarose resin.

Polyclonal antibodies from rabbits and chickens against the estrogen receptor and related peptides. Use in the affinity isolation of estrogen receptors and the retrieval of chromatin fragments associating with estrogen receptors.

Polyclonal antibodies from chickens and rabbits have been prepared against polypeptides representing two regions of the human estrogen receptor (hER). The estrogen receptor (ER) peptides used as antigens were overproduced in Escherichia coli. When indicated, the antibodies were affinity purified using resins to which the antigens contained in bacterial inclusion bodies had been coupled in high yield to epoxy-activated agarose. The antibodies recognize denatured human, bovine, rat, and rabbit ER in immunoblotting experiments. Immuno-precipitation of native ER protein was readily accomplished using rabbit antisera and immobilized protein A. The chicken antibodies, available in larger quantities, were also useful for immunoisolation after coupling to agarose. With the use of these reagents, the selective retrieval of chromatin fragments from MCF-7 cells that interact with ER has been achieved.

Effects of steroids on the synthesis and metabolism of phosphatidylethanol in phorbol ester-activated lymphocytes.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate activates the phospholipase D pathway in bovine lymphocytes, leading to a synthesis of phosphatidylethanol (PEt) from exogenous alcohol. Concomitant treatment of the cells with 10(-8) M etiocholanolone, dehydroepiandrosterone or 16 alpha-bromo-epiandrosterone results in the production of phosphatidylethanols that carry metabolically altered forms of arachidonic acid at position 2. The observed steroid response appears to be mediated by a receptor mechanism in that it depends on the synthesis of new RNA and protein. The spectrum of steroids producing the response suggests that the postulated receptor system may be distinct from the well-studied glucocorticoid, progesterone, estrogen and androgen specific receptors. The possible relevance of these new metabolites of ethanol to the problem of alcoholism in humans and to the field of carcinogenesis in general is discussed.

Antisera to a synthetic peptide recognize native and denatured rat estrogen receptors.

Specific polyclonal antisera to the rat estrogen receptor (rER) were developed using a synthetic peptide as the antigen. The peptide corresponds to amino acids 270-284 deduced from the cloned rER cDNA and has no homology to other steroid hormone receptors. Antipeptide antisera raised in rabbits specifically recognize a 67,000 mol wt protein, shown to be the rER, in Western blot experiments. In immunoprecipitation experiments, one tested antiserum bound unoccupied as well as 17 beta-estradiol-occupied rERs, indicating that this region is exposed in both receptor forms. This antiserum shows no cross-reactivity with rat progesterone or glucocorticoid receptors. Cross-reactivity with rabbit, human, and, to a lesser extent, bovine ERs was observed.

Isolation of proteoglycans using phenol extraction and isopycnic centrifugation in cesium trifluoroacetate.

A procedure for the rapid isolation of heparan sulfate proteoglycans from Zwittergent lysates of cultured cells and cetylpyridinium precipitates of culture medium is described. After removal of most nonglycosylated proteins by phenol extraction, the proteoglycans are fractionated and isolated by isopycnic sedimentation in cesium trifluoroacetate gradients. The density-isolated proteoglycans, which are largely free of high molecular weight DNA, RNA, and nonglycosylated proteins, are readily amenable to chemical and enzymatic characterization. These procedures, which are designed for efficient and rapid isolation of lipophilic proteoglycans, also appear applicable to the isolation of other high density proteoglycans.

Phorbol esters activate proteoglycan metabolism in human colon cancer cells en route to terminal differentiation.

Tumor-producing phorbol esters [e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA)] induce changes in a human colon cancer cell line, VACO 10MS, that mimic terminal differentiation: a rapid blockade of DNA replication and cell division, a marked increase in cell adhesion properties with striking changes in morphology, and the acquisition of ion-transporting activities. The present report shows that the triggering of this terminal differentiation sequence by TPA is associated with a rapid release of heparan sulfate proteoglycans from the cell surface that is soon followed by an acceleration of proteoglycan synthesis. The activation of the release mechanism is independent of ongoing protein synthesis, whereas the resynthesis of the proteoglycans requires the production of new proteins. A persistent high rate of proteoglycan synthesis and release appears correlated with the progression of the colon cell into the terminal differentiation state. Bryostatin 1, an agent which has been shown previously to block the TPA-induced terminal differentiation of this cell line, also largely prevents the TPA effects on proteoglycan metabolism. Since both TPA and bryostatin 1 produce their effects through the activation of members of the protein kinase C class of enzymes, it is proposed that the differentiation state of these colon cancer cells may be regulated by a differential activation of isozymes or a ligand-directed phosphorylation of proteins that are involved in proteoglycan metabolism.

Synthesis of phosphatidylethanol--a potential marker for adult males at risk for alcoholism.

Phosphatidylethanol, whose synthesis is catalyzed by a phospholipase D in a transphosphatidylation reaction, is a unique metabolite of ethanol. Phorbol 12-tetradecanoate 13-acetate, a tumor-promoting phorbol ester and stimulator of protein kinase C, activates this enzyme in peripheral blood lymphocytes. This system has been developed into an assay for measuring the potential of this pathway in human subjects. A pilot study of phosphatidylethanol synthesis in lymphocytes of adult males who have both an alcohol dependency and a family history of alcoholism has revealed that the average potential for phosphatidylethanol synthesis in this population is significantly elevated over that of control subjects.

12-O-tetradecanoylphorbol-13-acetate activates phosphatidylethanol and phosphatidylglycerol synthesis by phospholipase D in cell lysates.

A cell-free system for the synthesis of phosphatidylalcohols was developed in sonicates of HL-60 cells. With [32P]phosphatidylcholine as the exogenous substrate, both phosphatidylethanol and phosphatidylglycerol were formed through a phospholipase D-catalyzed transphosphatidylation of ethanol and glycerol, respectively. The transphosphatidylation by phospholipase D was stimulated in vitro by 12-O-tetradecanoylphorbol-13-acetate (TPA) and required the addition of ATP for an optimal response. GTP-gamma-S, an activator of G protein systems, also stimulated the process by an independent mechanism. It is postulated that the stimulation of phospholipid metabolism through phospholipase D activation represents an important mechanism whereby TPA might modulate intracellular signal generating systems or influence the activity of membrane-bound proteins by altering their lipid environment.

Bryostatin 1 antagonizes the terminal differentiating action of 12-O-tetradecanoylphorbol-13-acetate in a human colon cancer cell.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a highly active representative of the tumor-promoting phorbol esters, induces a rapid terminal differentiation of a human colon cancer cell line. Bryostatin 1, a macrocyclic lactone, completely counteracts this effect of TPA and promotes continued replication. The observed responses provide a system for identifying cellular processes which are involved in the induced terminal differentiation of human colon cancer cells.

Hemin inhibits virion-associated reverse transcriptase of murine leukemia virus.

The virion-associated reverse transcriptase activity of Rauscher murine leukemia virus was inhibited by freshly prepared hemin at a concentration of 10(-4) M. When the hemin solution was aged at room temperature for 5 days, the concentration of 50% inhibition decreased to as low as 10(-7) M. Removal of O2 from the solution partially prevented the aging. The hemin inhibition was reversible and appears to be directed against the enzyme rather than the template. Hemin did not inhibit the activity of reverse transcriptase purified from avian myeloblastosis virus.