Characterization of the rat connexin40 promoter: two Sp1/Sp3 binding sites contribute to transcriptional activation.

OBJECTIVES: The gap junction protein connexin40 (Cx40) is differentially expressed during embryonic development and in adult tissues, for which the molecular basis is unknown. In order to elucidate the molecular mechanisms controlling Cx40 expression, we set out to map and characterize its promoter. METHODS: The transcriptional activity of individual rat Cx40 (rCx40)-derived promoter fragments fused to the luciferase reporter gene was determined by transfection/reporter assays in Cx40-expressing (A7r5, rat smooth muscle embryonic thoracic aorta cells, and BWEM, v-myc transformed rat fetal cardiomyocytes) and Cx40-nonexpressing cells (N2A, mouse neuroblastoma cells). The nature of DNA-protein interactions was investigated by a combination of standard electrophoretic-mobility-shift assays (EMSA) and EMSA/antibody supershift assays. RESULTS: Quantification of luciferase activity in cell lysates revealed that a 235-base-pair fragment, in between map positions -150 and +85 relative to the transcription initiation site, is able to provide for a significant level of transcription in both Cx40-expressing (A7r5, BWEM) and -nonexpressing (N2A) cells. These results indicate that this region contains the basal promoter but is not sufficient to completely determine the endogenous Cx40-expression pattern within these cell types. In search for the responsible transcriptional regulatory element(s), additional segments of the (-150, +85) region were deleted and the remaining fragments were tested for transcriptional activity. These studies established that the regions in between map positions (-96, -71) and (+58, +85) contribute to promoter activity. EMSA with these regions revealed that predominantly two DNA-protein complexes are formed upon incubation with either A7r5, BWEM or N2A nuclear extracts, which could be both inhibited by including excess oligonucleotide containing the Sp1 consensus binding site in the binding reaction. Purified recombinant human Sp1 provided also for a shift in the EMSA using these promoter regions as target fragments. When the DNA-protein complexes formed with nuclear extract were subsequently incubated with either an anti-Sp1 or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, indicating Sp1 and Sp3 binding to both the (-98, -64) and (+53, +87) regions. The introduction of mutations within the core sequence of the putative Sp1/Sp3 binding sites present in these regulatory elements reduced the level of transcriptional activity and abrogated Sp1/Sp3 binding to these sites. CONCLUSION: The results indicate that at least two Sp1/Sp3 binding sites in the rCx40 promoter contribute to the transcriptional activation of its gene in cultured cells.

Human SCN5A gene mutations alter cardiac sodium channel kinetics and are associated with the Brugada syndrome.

BACKGROUND: Primary dysrhythmias other than those associated with the long QT syndrome, are increasingly recognized. One of these are represented by patients with a history of resuscitation from cardiac arrest but without any structural heart disease. These patients exhibit a distinct electrocardiographic (ECG) pattern consisting of a persistent ST-segment elevation in the right precordial leads often but not always accompanied by a right bundle branch block (Brugada syndrome). This syndrome is associated with a high mortality rate and has been shown to display familial occurrence. METHODS AND RESULTS: Pharmacological sodium channel blockade elicits or worsens the electrocardiographic features associated with this syndrome. Hence, a candidate gene approach directed towards SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel, was followed in six affected individuals. In two patients missense mutations were identified in the coding region of the gene: R1512W in the DIII-DIV cytoplasmic linker and A1924T in the C-terminal cytoplasmic domain. In two other patients mutations were detected near intron/exon junctions. To assess the functional consequences of the R1512W and A1924T mutations, wild-type and mutant sodium channel proteins were expressed in Xenopus oocytes. Both missense mutations affected channel function, most notably a 4-5 mV negative voltage shift of the steady-state activation and inactivation curves in R1512W and a 9 mV negative voltage shift of the steady-state activation curve in A1924T, measured at 22 degrees C. Recovery from inactivation was slightly prolonged for R1512W channels. The time dependent kinetics of activation and inactivation at -20 mV were not significantly affected by either mutation. CONCLUSIONS: Two SCN5A mutations associated with the Brugada syndrome, significantly affect cardiac sodium channel characteristics. The alterations seem to be associated with an increase in inward sodium current during the action potential upstroke.

Genomic organization of the rat connexin40 gene: identical transcription start sites in heart and lung.

OBJECTIVES: The gap junction protein connexin(Cx)40 is developmentally and tissue-specifically expressed. How Cx40 expression is regulated is unknown. We therefore set out to characterize the 5'-untranslated end of both the Cx40 gene and mRNA from different tissues and ages and to identify the Cx40 promoter region. METHODS: The PCR method 5'-RACE was used to amplify the 5'-end of rat Cx40 mRNAs. Genomic rat Cx40 clones were isolated from a lambda EMBL3 library. The promoter sequence was isolated by long distance PCR. The transcription start site was identified by primer extension and RNase protection assays. RESULTS: Comparison of Cx40 genomic DNA and mRNA sequences revealed that the Cx40 gene contains a small untranslated exon, exon I, which is separated from the coding sequences by an intron of at least 5.5 Kb. The untranslated 5'-end of Cx40 mRNA sequences from adult rat lung, neonatal and adult rat heart and the rat aortic smooth muscle cell line A7r5 were identical. While the same transcription start site was found for the Cx40 mRNAs from different tissues and ages, and amount of Cx40 mRNA differed between tissues as follows: A7r5 cells > neonatal lung > adult lung > or = neonatal atrium > neonatal ventricle; Cx40 mRNA from adult atrium and ventricle was not readily detected by primer extension and RNase protection analyses. The genomic sequence upstream of the transcription start site contains multiple consensus binding sites for transcription factors putatively responsible for spatio-temporal control of Cx40 gene expression. CONCLUSIONS: Similar to other connexin genes, the Cx40 gene contains two exons. The same exon I sequence is present in all tissues and developmental stages examined and the relative amounts of Cx40 mRNA in these compare well with published data. Together our data suggest that tissue-specific and developmentally regulated expression of the Cx40 gene is controlled within the same promoter region by mechanisms that have yet to be detailed.

Altered pattern of connexin40 distribution in persistent atrial fibrillation in the goat.

INTRODUCTION: Since altered expression of gap junction proteins (connexins) in diseased myocardial tissue may lead to abnormal electrical coupling between cardiomyocytes and hence contribute to arrhythmogenesis, the expression of connexin(Cx)40 and Cx43 was studied in atrial appendage from goats in sinus rhythm (SR) and persistent atrial fibrillation (AF). METHODS AND RESULTS: Biopsies were taken from the left and right atrial appendages from goats in SR or after pacing-induced persistent AF. Analyses of Cx40 and Cx43 mRNA and protein levels, using quantitative (competitive) polymerase chain reaction and western blotting, respectively, revealed no significant changes in the overall expression of Cx40 and Cx43 as a result of persistent AF. At the cellular level, immunohistochemistry and confocal laser scanning microscopy showed a homogeneous distribution of either connexin in atrial sections taken during SR. After induction of AF, the distribution of Cx43 gap junctions was unchanged whereas the Cx40 pattern showed marked inhomogeneities with small areas (0.15 to 0.6 mm in diameter, 25% of section surface area) of low-density Cx40 located between larger areas of normal (unchanged) Cx40 density. Activation mapping (244 electrodes, spatial resolution 2.25 mm) of the right atrial wall did not reveal changes in atrial conduction velocity. CONCLUSION: Pacing-induced persistent AF in the goat gave rise to changes in the spatial organization of Cx40 gap junctions. Although the overall conduction velocity appeared not to have changed, microheterogeneities in conduction due to the local redistribution of Cx40 gap junctions might have contributed to the initiation and maintenance of AF.

Dysbetalipoproteinemia in a kindred with hypobetalipoproteinemia due to mutations in the genes for ApoB (ApoB-70.5) and ApoE (ApoE2).

We identified the first insertion mutation that specifies an apolipoprotein (apo)B truncation, apoB-70.5, in a father and son with hypobetalipoproteinemia (total and low-density lipoprotein [LDL] cholesterol < 5th percentile, plasma apoB levels approximately one third of normal). The mutation is due to insertion of an adenine (A) into a 7-A repeat between cDNA position 9754 and 9760 of the apoB gene, resulting in a frame shift of 13 new amino acids and a termination codon at amino acid residue 3197. The DNA mutation cosegregated with the apoB truncation and hypobetalipoproteinemia in the kindred. The two apoB-70.5/apoB-100 heterozygotes also are apoE2 homozygotes by genotyping; beta-very-low-density lipoprotein (VLDL) was present, and VLDL cholesterol/triglyceride ratios were increased (0.29) in the plasmas of both. Density gradient ultracentrifugation and gel filtration chromatography profiles showed increased amounts of particles in the VLDL and intermediate-density lipoprotein density and size ranges and relatively smaller peaks of LDL than in controls. Two populations of LDL were present, ApoB-70.5 was primarily associated with LDL particles of higher density and of smaller size than the LDL particles containing apoB-100. ApoB-48-containing particles were present in the VLDL of fasting plasmas of both subjects, and the postprandial levels of chylomicrons and remnants as measured by the vitamin A fat tolerance test were increased. In conclusion, both subjects heterozygous for apoB-70.5 and homozygous for apoE2 showed the classic characteristics of dysbetalipoproteinemia superimposed onto the hypolipoproteinemia state.

Apolipoprotein B-38.9 does not associate with apo[a] and forms two distinct HDL density particle populations that are larger than HDL.

We have identified a new truncated apolipoprotein B (apoB) that provides insights into the interaction of apoB with apo[a] and with lipids. Both total and LDL-cholesterol were below the 5th percentile in the proband; Lp[a] was 28 mg/dl. Four other affected individuals were identified in this kindred. Immunoblotting of plasma apoB-containing lipoproteins with an anti-apoB monoclonal antibody revealed a major band for apoB-100 and a minor band with apparent M(r) 217 kDa. The apoB truncation is due to a -1 frameshift mutation, consisting of a cytosine deletion at cDNA position 5444, that results in the translation of 22 novel amino acids terminating at residue 1767. The mutation was confirmed in the affected subjects by allele-specific oligonucleotide (ASO) analysis. Gel filtration of whole plasma revealed that the minority of apoB-38.9 eluted with IDL- and LDL-sized particles, while the majority (approximately 60%) eluted between LDL and HDL. Lp[a] eluted between VLDL and LDL. Upon preparative density gradient ultracentrifugation (DGUC), the majority of the plasma apoB-38.9 (approximately 65%) floated at a density of 1.12 g/ml coincident with the major peak of HDL cholesterol. Lp[a] floated at a peak density of 1.08 g/ml between LDL and HDL. Immunoblots of the apoB-38.9-containing HDL density DGUC fractions subjected to nondenaturing gradient gel electrophoresis (GGE) demonstrated two apoB-38.9-containing particle populations with diameters of approximately 15 nm and approximately 18 nm, respectively. Lipoproteins of these sizes were also detected when whole plasma was subjected to GGE and immunoblotting. The 15-18 nm lipoproteins correspond to the gel filtration populations eluting between LDL and HDL. Lysine-Sepharose chromatography of plasma yielded retained products that contained apo[a] and apoB-100 but not apoB-38.9. Immunoprecipitation of whole plasma with monospecific polyclonal anti-human apo[a] showed apo[a] and apoB-100, but no apoB-38.9 to be present in precipitates. ApoB-100 and apoB-38.9 were present in supernates. In in vitro incubations, recombinant apo[a] formed complexes with apoB-100 but not with apoB-38.9-containing particles. Our results show that the apoB-38.9 protein can be found in a variety of lipoproteins; however, the majority of apoB-38.9-containing lipoproteins float at a density equivalent to HDL but are larger than HDL, being intermediate in size between apoB-100 LDL and HDL. The heterogeneity of apoB-38.9 lipoproteins may reflect their dual tissue source, i.e., liver and intestine, and the discordance between size and density indicates a disproportionately reduced association of lipids with apoB-38.9. Finally, our data suggest that apoB-38.9 is incapable of forming complexes with apo[a] in plasma.

Apolipoprotein B-52 mutation associated with hypobetalipoproteinemia is compatible with a misaligned pairing deletion mechanism.

We have identified a new truncation of apoB in a large kindred with hypobetalipoproteinemia that arose by an ambiguous deletion of one of four different groups of base-pairs. Eleven affected members of the kindred had total cholesterols (C) of 114 +/- 28, LDL-Cs of 46 +/- 21, and apoBs of 47 +/- 25 (all in mg/dl, mean +/- SD). These levels were lower (P < 0.0001) than in 15 unaffected relatives. On Western blotting, apoB-100 and a second major band corresponding to apoB-52 were seen in the affected individuals. The majority of the plasma apoB-52 was associated with a smaller than normal low density lipoprotein (LDL) particle. The molecular basis for this apoB-52 truncation is a 5-bp deletion, converting the sequence between cDNA nucleotide 7276 and 7283 from 5'-AAGTTAAG-3' into the mutant sequence 5'-AAG-3'. This results in a frameshift starting at amino acid residue 2357 and a termination codon at amino acid residue 2362. Deletion of one of four different groups of five consecutive bases, i.e., AAGTT, AGTTA, GTTAA, and TTAAG, all result in the same mutant sequence. Thus, the precise deletion is ambiguous. We propose that a misaligned pairing mechanism involving repeat sequences is compatible with this deletion mutation. We have noted similar ambiguous deletions associated with apoB-37, apoB-40, and a number of single base deletions and some may also be explained by a misaligned pairing mechanism. Small ambiguous deletions appear to constitute a major proportion of the apoB gene mutation spectrum suggesting that it may be a suitable model for studying the mechanisms of such mutations.

A comparison of the effects of an extract of feverfew and parthenolide, a component of feverfew, on human platelet activity in-vitro.

Extracts of the herb feverfew inhibit human blood platelet aggregation and secretion induced by a number of agents in-vitro and this may relate to the beneficial effects of feverfew in migraine. We previously identified several compounds with antisecretory activity in human blood platelets using adrenaline as the stimulant. In the present study, we have compared the inhibitory activity of one of these compounds, parthenolide, with that of crude feverfew extract. The effects of both on [14C]5-HT secretion from platelets and on platelet aggregation induced by a number of different stimulants were determined. The activating agents studied included the phorbol ester PMA, ADP, arachidonic acid, collagen, the thromboxane mimetic U46619, the calcium ionophore A23187, the diacylglycerol analogue OAG and adrenaline. The results show that there are marked similarities between the effects of feverfew extract and of parthenolide on both [14C]5-HT secretion and platelet aggregation, which is consistent with the effects of feverfew extract on platelets being brought about by parthenolide or similar compounds in the extract. Only in one case, when A23187 was used as the stimulatory agent, was there any discrepancy, which may have been due to materials in the extract other than parthenolide. Both feverfew extract and parthenolide were more effective as inhibitors of the [14C]5-HT secretion and aggregation induced by some agents and not others, and were most effective as inhibitors of the [14C]5-HT secretion (but not the aggregation) induced by PMA. This suggests that the effects of feverfew/parthenolide on the protein kinase C pathway warrants further study.

Effects of an extract of feverfew on endothelial cell integrity and on cAMP in rabbit perfused aorta.

Extracts of feverfew inhibit platelet aggregation and secretion of granular contents from platelets and other cells. They also modify the interaction of platelets with collagen substrates: feverfew extracts inhibit both platelet spreading and formation of thrombus-like platelet aggregates on the collagen surface. We have now investigated the effect of an extract of feverfew on the vessel wall using rabbit aortas that were perfused with a physiological salt solution in-situ. Addition of feverfew extract to the perfusion medium protected the endothelial cell monolayer from perfusion-induced injury and led to a reversible increase in the cAMP content of aorta segments. The results indicate that feverfew may have a vasoprotective effect in addition to its effects on platelets.

Effects of an extract of feverfew (Tanacetum parthenium) on arachidonic acid metabolism in human blood platelets.

It has been suggested that feverfew extracts (FE) inhibit platelet behaviour via effects on platelet sulphydryl (SH) groups. In the present study we found evidence that FE inhibits uptake as well as liberation of arachidonic acid (AA) into/from platelet membrane phospholipids (PL).

Inhibition of platelet behaviour by feverfew: a mechanism of action involving sulphydryl groups.

Extracts of feverfew inhibit platelet aggregation and the platelet release reaction. The active components are believed to be sesquiterpene lactones such as parthenolide. Evidence is presented that inhibition of platelet behaviour is via neutralization of sulphydryl groups either inside or outside the cell. The precise nature of the sulphydryl groups that are susceptible to feverfew and are involved in platelet aggregation and the release reaction have not yet been defined.

Feverfew--an antithrombotic drug?

The effects of an extract of the plant feverfew on the interaction of platelets with surfaces coated with human collagens of type III and IV (CIII, CIV), and on the integrity of the endothelial cell (EC) monolayer in perfused rabbit aorta were studied. It was shown that feverfew extract (FE) inhibited the deposition of [51Cr]-labelled platelets on both CIII and CIV in a dose-dependent way. Similar concentrations of FE were needed to inhibit formation of surface-bound aggregates in CIII and platelet spreading on CIV in both platelet-rich plasma and GFP. When aorta segments were perfused in situ with a physiological salt solution, the addition of FE to the solution protected the EC monolayer from spontaneous injury. The results indicate that feverfew may have antithrombotic potential in addition to its claimed benefit in fever, migraine and arthritis.

An extract of feverfew inhibits interactions of human platelets with collagen substrates.

The interaction of platelets with surfaces coated with collagens of type III (C III) or IV (C IV) has been studied by measuring the deposition of 51-Cr-labeled platelets and by scanning electron microscopy (SEM). Experiments were performed using platelet-rich plasma (PRP) and suspensions of gel-filtered platelets (GFP). Platelets were deposited on C III mainly as surface-bound aggregates. In contrast they were deposited on C IV mainly as spread forms of individual cells. Formation of aggregates on C III was more extensive for PRP than for GFP; in contrast platelet spreading on C IV was more extensive for GFP than for PRP. The effects of an extract of the plant feverfew on platelet-collagen interactions were determined. Feverfew extract inhibited the deposition of 51-Cr-labeled platelets on both C III and C IV in a dose-dependent way. Similar concentrations of extract were needed to inhibit the formation of surface-bound aggregates and to inhibit platelet spreading in both PRP and GFP.

Extracts of feverfew may inhibit platelet behaviour via neutralization of sulphydryl groups.

It has been suggested that extracts of feverfew may inhibit platelet behaviour via effects on platelet sulphydryl groups. In the present study we have obtained evidence for such a mode of action. Compounds that contain sulphydryl groups such as cysteine and N-(2-mercaptopropionyl)glycine prevented the inhibition of platelet behaviour by feverfew. Feverfew and parthenolide (one of the active components of feverfew) dramatically reduced the number of acid-soluble sulphydryl groups in platelets. This effect occurred at concentrations similar to those that inhibited platelet secretory activity. Feverfew itself did not induce the formation of disulphide-linked protein polymers in platelets but polymer formation occurred when aggregating agents were added to feverfew-treated platelets. Feverfew evoked changes in the metabolism of arachidonic acid that were similar to those observed in glutathione-depleted platelets.

Compounds extracted from feverfew that have anti-secretory activity contain an alpha-methylene butyrolactone unit.

Extracts of feverfew inhibit secretion of granular contents from platelets and neutrophils and this may be relevant to the therapeutic value of feverfew in migraine and other conditions. In this investigation we fractionated an extract of feverfew and obtained eleven fractions with antisecretory activity. The activity. The active fractions, together with two fractions that were devoid of anti-secretory activity, were examined using 1H NMR and infrared spectroscopy. All the active fractions (but neither of the inactive fractions) contained compounds with an alpha-methylene butyrolactone unit. Five compounds that contain this unit were identified as parthenolide, 3-beta-hydroxyparthenolide, secotanapartholide A, canin and artecanin, all of which are sesquiterpene lactones. It is very likely that these and other sesquiterpene lactones that contain an alpha-methylene butyrolactone unit are responsible for the anti-secretory activity in extracts of feverfew.