Graded contrast echocardiography in pulmonary arteriovenous malformations.

To compare the results of transthoracic contrast echocardiography (TTCE) adding a grading scale with the results of thoracic computed tomography (CT) in order to optimise the use of both techniques. 95 patients with hereditary haemorrhagic telangiectasia (HHT) were examined with TTCE and thoracic CT to detect pulmonary arteriovenous malformations (PAVMs). According to previous studies, TTCE was divided into a four grade scale depending on the degree of opacification of the left ventricle after the administration of a contrast agent. Of the 95 patients (50.5% female; mean age 46 yrs), none with normal or grade 1 TTCE had detectable PAVMs on thoracic CT. Shunts of grades 2, 3 and 4 were associated with PAVMs according to thoracic CT in 25, 80, and 100% of the cases. There was a statistically significant association between the TTCE grade and the detection of a PAVM by thoracic CT. There were also statistically significant associations between TTCE grade and the cardiac cycle when the contrast was first visible in the left atrium, and size of the feeding artery. Graded TTCE and timing of left atrium opacification may be useful techniques in selecting HHT patients for PAVM screening with thoracic CT scans.

The ß2-adrenergic receptor antagonist ICI-118,551 blocks the constitutively activated HIF signalling in hemangioblastomas from von Hippel-Lindau disease.

One of the major consequences of the lack of a functional VHL protein in von Hippel-Lindau disease, a rare cancer, is the constitutive activation of the HIF pathway. This activation ends up in the generation of Central Nervous System (CNS) Hemangioblastomas among other tumours along the lifespan of the patient. Nowadays, only surgery has been proven efficient as therapy since the systemic attempts have failed. Propranolol, a non-specific ß1-and ß2-adrenergic receptor antagonist, was recently designated as the first therapeutic (orphan) drug for VHL disease. Nevertheless, its ß1 affinity provokes the decrease in blood pressure, being not recommended for low or regular blood pressure VHL patients. In order to overcome the ß1-drawback, the properties of a high specific ß2-adrenergic receptor blocker named ICI-118,551 have been studied. ICI-118,551 was able to decrease Hemangioblastomas cell viability in a specific manner, by triggering apoptosis. Moreover, ICI-118,551 also impaired the nuclear internalization of HIF-1α in Hemangioblastomas and hypoxic primary endothelial cells, reducing significantly the activation of HIF-target genes and halting the tumour-related angiogenic processes. In this work, we demonstrate the therapeutical properties of ICI-118,551 in VHL-derived CNS-Hemangioblastoma primary cultures, becoming a promising drug for VHL disease and other HIF-related diseases.

Cloning and characterization of the scute (sc) gene of Drosophila subobscura.

The scute (sc) gene, a member of the achaete-scute complex of Drosophila melanogaster, has dual functions: sisterless (sis-b) function required for sex determination and dosage compensation and scute function, which is involved in neurogenesis. The sc homologue of D. subobscura was cloned. It lacks introns and encodes a single 1.7-kb transcript slightly larger than that of D. melanogaster (1.6 kb). The sc protein of D. subobscura is slightly larger than that of D. melanogaster (382 vs. 345 amino acids). Sequence comparisons between both species show the Sc protein to have a highly conserved bHLH domain. Outside this domain, amino acid replacements are not randomly distributed. Two additional conserved domains, of 20 and 36 amino acids, are present near the C-terminal end. They may represent domains confering specificity upon the Sc protein with respect to other proteins of the achaete-scute complex. In its 3' untranslated region, Sc RNA contains uridine stretches, putative Sxl protein DNA-binding sites. The D. subobscura Sc protein can cooperate with other D. melanogaster bHLH proteins because D. subobscura sc supplies sis-b function when introduced into D. melanogaster transgenic flies mutant for sc.

Atorvastatin-induced endothelial nitric oxide synthase expression in endothelial cells is mediated by endoglin.

Endoglin, a transforming growth factor ß (TGF-ß) receptor type III, is co-expressed with endothelial nitric oxide synthase (eNOS) in aortic endothelium in atherosclerotic plaques of mice. Interestingly, atorvastatin (ATV) is able to increase both endoglin and eNOS expression and reduce plaque size beyond its lipid lowering effects but by unknown mechanisms. We hypothesized whether inflammation modulates ATV-dependent induction of endoglin and eNOS expression in vitro in endothelial cells and whether ATV-induced eNOS expression is regulated via endoglin. After treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α, endoglin and eNOS protein expression was reduced, concomitantly with increased levels of cell surface VCAM-1 and soluble endoglin, as determined by flow cytometry, Western blot and ELISA analyses. By contrast, ATV treatment increased endoglin and eNOS protein expression, while preventing TNF-α-mediated downregulation of endoglin and eNOS protein levels. Moreover, suppression of endoglin using small interfering RNA (siRNA), but not inhibition of TGF-ß signaling with SB431542, abrogated ATV-induced eNOS expression. These results suggest that ATV treatment prevents inflammation-reduced endoglin and eNOS expression in endothelial cells and that ATV-induced eNOS expression strongly depends on the proper expression of endoglin in HUVECs. Possible implications of these findings might be reflected in pathological conditions characterized by reduced expression of endoglin and eNOS as for example in hereditary hemorrhagic telangiectasia or in other endothelial dysfunctions.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana.

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

Organophosphorous pesticides in breast cancer progression.

Environmental substances may be involved in the etiology of breast cancers. Many studies have found an association between cancer in humans and exposure to agricultural pesticides. Organophosphorous pesticides have been used to control mosquito plagues. Parathion and malathion, organophosphorous pesticides are cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. Their primary target of action in insects is the nervous system whereby they inhibit the enzyme acetylcholinesterase at synaptic junction. Atropine is a parasympatholytic alkaloid used as an antidote to acetylcholinesterase inhibitors. We have established an experimental breast cancer model, where epithelial cells in the rat mammary gland underwent a stepwise transformation into malignant cells by exposure to pesticides (Cabello et al, 2001). The aim of this work was to examine whether pesticides were able to induce progression of malignant transformation of a human breast epithelial cell line, MCF7. These results showed that parathion and malathion increased PCNA and induced mutant p53 protein expression of MCF7 cells in comparison to controls and atropine inhibited such action. These results indicated that organophosphorous pesticides can induce more changes in this malignant breast cell line, inducing another step in the progression of the transformation process and atropine on the other hand inhibited the effect of such substances.

The Balbiani ring 6 induction in Chironomus.

Balbiani ring (BR) genes in polytene chromosomes of Chironomus salivary glands code for secretory proteins of the sp-I family, 10(6) D. They are used by the aquatic larva to spin a housing and feeding tube. The expression of the BR gene family undergoes correlated changes depending on the environment. In the presence of certain sugars, ethanol or glycerol the normally most active BR2 regresses and its products disappear. There is a parallel induction of a new BR, BR6, and a new sp-I protein. The change seems to represent an adaptative response to phosphate depletion in the larval haemolymph produced by the inducing agents. The BR2 (and BR1) products are heavily phosphorylated and the BR6 product non-phosphorylated. One of the BR2-coded sp-I proteins is cleaved off close to the C-terminus and material with properties expected of the resulting polypeptide can be recovered in the nuclei, accumulating in the BR. This might represent a feed-back signal from translation to transcription.

[Effect of beta-ecdysterone in competitive cultures of Drosophila melanogaster]. / Efecto de la beta-ecdisterona en cultivos en competencia de Drosophila melanogaster.

High levels of beta-ecdysterone (more than 1 microgram/ml) have been shown to decrease larva-adult viability and to speed up developmental times when supplied to the media in crowding cultures. The highest doses (100 micrograms/ml) of ecdysterone suppresses almost completely the phenomenon of larval stop in the third instar of development, first reported by our laboratory in overcrowded situations. Thus, one may deduce that stopped larvae could have low levels of ecdysone, and perhaps these are the ultimate physiological cause of their arrested development before the critical larva-pupa molt.

The C-terminal DNA-binding domain of Chironomus BR gene products shows preferential affinity for (dA.dT)-rich sequences.

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midge Chironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived from BR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the large BRs (BR1 and BR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of the BR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of the BR genes.

Structural constraints in expansion segments from a midge 26S rDNA.

DNA sequences representing approximately 40% of the large-subunit rRNA gene from the lower dipteran Chironomus thummi were analyzed. Once aligned with their Drosophila counterparts, sequence and base content comparisons were carried out. Sequence identity was found to be high overall, except for six regions that displayed a local bias in nucleotide composition toward AT. These regions were identified as expansion segments D3, D4, D5, D6, D7a, and D12. Besides base sequence divergence, differences in length were observed between the respective variable domains of the two species, particularly for D7a. Prediction of secondary structure showed that the folding of the Chironomus expansion segments analyzed is in agreement with the general patterns proposed for eukaryotic LSU rRNA. The comparison with Drosophila revealed also that the Chironomus secondary structures of the variable domains are supported by multiple compensatory substitutions or even compensatory insertions. Chironomus D7a displayed an unusual structural feature with respect to the insect D7a models that have been inferred up to now. The structural constraint observed in the expansion segments of Diptera so distantly related as midges and Drosophila suggests that these regions contribute to some functional role. Concerning the D7a of insects so far analyzed, there can be, in addition to a conserved secondary structure, a nucleotide composition constraint that might be important for the process giving rise to the alpha and beta halves of the 26S rRNA.

Functional analysis of the 3'-terminal part of the Balbiani ring 2.2 gene by interspecies sequence comparison.

An interspecies comparison was made between the 3' ends of Balbiani ring genes from Chironomus. The comparison was focused on the BR2.2 gene, and a part at the 3' end from Chironomus pallidivittatus (which included also a segment of the gene core) was cloned. Its sequence, and other previously published BR sequences from this species and from Chironomus tentans were used in the analysis. The 3' parts of these repetitive genes can be divided into a region belonging to the core of the genes followed by a terminal region. In the core region the repeats (each of which consists of a constant part and a subrepeated part) are highly similar and the constant parts show little interspecies differentiation. Furthermore, the two parts of the repeats are units in an evolutionary and probably also functional sense. The terminal region contains modified constant units, usually isolated between acidic so-called cys regions, the whole arrangement lying upstream of an intron toward a 3'-terminal exon. Most of the modified constant units are mosaics in rates of evolution with stable outer quarters bordering to equally stable cys regions and a central half with a very high rate of evolution. One of the terminal units, present only in the BR2.2 gene and second from the end, differs distinctly not only from corresponding core units but also from other terminal units in the three normally active BR genes. It lies upstream of all cys regions and is evolutionarily conserved over most of its length. Furthermore, two-dimensional protein structure prediction does not exclude an endoproteolytic cleavage site in this unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat shock activation of telomeric sequences in different tissues of Chironomus thummi.

The heat shock transcription factor (HSF) has been found by immunocytochemistry using the Drosophila HSF antibody at T-BRIII, a telomeric heat shock-induced puff in polytene chromosomes of Chironomus thummi salivary glands. Other heat shock-activated loci were also positively stained by the antibody. Neither the telomeres nor other heat shock loci were labeled under control conditions. These results support the presence of a heat shock gene at T-BRIII despite its peculiar location and molecular organization, different from other well-characterized heat shock genes in Diptera. This locus is similarly induced and transcribed under heat shock in Malpighian tubules, another larval polytenic tissue. Transcription from telomeric-associated sequences has also been found in control polytenic and diploid tissues. The meaning of transcription and heat shock activation of telomeric sequences is discussed in relation to the organization of telomeres and compared to possible equivalents in other known heat shock loci.

Heat-shock induction and cytoplasmic localization of transcripts from telomeric-associated sequences in Chironomus thummi.

Transcription of telomeric-associated sequences has been detected in the salivary gland cells of the larvae Chironomus thummi. In this species, a heat shock induces puffing at some telomeres, especially at one of the telomeres of chromosome III. We found that this process was concomitant with an increase in the overall telomeric transcript levels. Transcription was also observed in all the telomeres under control conditions, by in situ hybridization, even when these telomeres appeared to be in a nonpuffed state. The telomeric transcripts were found in both, the nuclei and, at higher levels, in the cytoplasmic extracts of salivary gland cells. The heat-shock activation, however, appeared to be restricted to the nuclear level. Telomeric transcription and the peculiar behavior of C. thummi telomeres after a heat shock are discussed.

Immunological detection of phenylalanine hydroxylase protein in Drosophila melanogaster.

A monoclonal antibody raised against monkey liver phenylalanine hydroxylase (PAH) has been used to detect this protein in Drosophila melanogaster. A cross-reacting material (CRM) band of apparent molecular mass 50-52 kDa, equivalent to that deduced for the Drosophila melanogaster PAH protein based on the pah gene cDNA sequence, has been detected. This CRM was analysed throughout development and showed an equivalent pattern to that reported for PAH activity in this insect, with maxima at pupariation and at pharate adult formation. Distribution of this CRM in larval tissues, the haemolymph and the adult body is mainly restricted to the larval fat body and the adult head. Demonstration of this CRM as the PAH protein comes from the correlation between the decreased PAH enzyme activities of two mutant strains and their decreased amounts of CRM by Western blotting.

Ku-related antigens are associated with transcriptionally active loci in Chironomus polytene chromosomes.

Antigens of Chironomus reactive with human sera containing anti-Ku antibodies and also with specific antibodies to each Ku subunit were characterized by immunoblot analysis. Three main antigen species were identified in nuclear-enriched extracts from salivary gland cells of Chironomus thummi, ranging in Mr from 55000 to 67000. The nuclear localization of Ku-related antigens in the dipteran Chironomus was studied by immunofluorescent labeling in polytene chromosomes of the salivary glands. Balbiani rings, loci highly active in transcription, were found to be strongly labeled by anti-Ku antibodies. Sugar-induced changes in the activity of the Balbiani ring genes were accompanied by the redistribution of Ku-related antigens as visualized by their absence in regressed Balbiani ring loci, and continued presence only in those that were transcriptionally active. A drastic change in the distribution of Ku-related antigens was also observed when C. thummi larvae underwent heat treatment as the immunofluorescent staining was restricted to previously described heat shock puffs. Anti-Ku sera reacted in addition with several chromosomal bands in which the presence of RNA polymerase II was also immunologically detected. The results show that Chironomus antigens reactive with anti-Ku antibodies are related to transcription in polytene chromosomes.

Synergistic cooperation between hypoxia and transforming growth factor-beta pathways on human vascular endothelial growth factor gene expression.

Signaling by transforming growth factor (TGF)-beta family members is mediated by Smad proteins that regulate gene transcription through functional cooperativity and association with other DNA-binding proteins. The hypoxia-inducible factor (HIF)-1 is a transcriptional complex that plays a key role in oxygen-regulated gene expression. We demonstrate that hypoxia and TGF-beta cooperate in the induction of the promoter activity of vascular endothelial growth factor (VEGF), which is a major stimulus in the promotion of angiogenesis. This cooperation has been mapped on the human VEGF promoter within a region at -1006 to -954 that contains functional DNA-binding sequences for HIF-1 and Smads. Optimal HIF-1alpha-dependent induction of the VEGF promoter was obtained in the presence of Smad3, suggesting an interaction between these proteins. Consistent with this, co-immunoprecipitation experiments revealed that HIF-1alpha physically associates with Smad3. These results demonstrate that both TGF-beta and hypoxia signaling pathways can synergize in the regulation of VEGF gene expression at the transcriptional level.

Identification of the spI products of Balbiani ring genes in Chironomus thummi.

The spI fraction of high molecular weight secretory proteins was analysed in Chironomus thummi. These proteins are encoded by giant Balbiani ring (BR) genes which develop specifically in salivary gland cells. Each component of the spI fraction was studied electrophoretically from early and middle 4th instar larvae and prepupae, as well from galactose-treated larvae where changes in the relative puffing pattern of BR1 and BR2 are known to occur. The spI fraction consists of at least two bands with electrophoretic mobilities slower than those of the spI components of Camptochironomus. The slow migrating component remains throughout the 4th larval instar, while the amount of the faster component changes, being abundant in early 4th instar and prepupae, but not present (or very weak) in middle 4th instar. The correlated shifts in BR puffing pattern during these developmental stages suggest that the slow and fast components are encoded by BR2 and BR1. The spI fraction is modified by galactose treatment, the fast component being induced in parallel with a decrease in the slow component. These changes are correlated with changes in the steady-state levels of RNA: an increase in BR1 RNA and a decrease in BR2 RNA, and of proteins. These proteins could correspond to the spIb and spIa fractions allocated to BR2 and BR1, respectively, in Camptochironomus. After galactose treatment a new faster band sometimes appears, that could correspond to the spIc fraction of Camptochironomus. A possible spId equivalent was also identified. In conclusion the main features of the spI family in C. thummi are similar to those of spI in Camptochironomus.