[Dispersion of QT interval in arterial hypertension with left ventricular hypertrophy]. / Dispersione dell'intervallo QT nell'ipertensione arteriosa con ipertrofia ventricolare sinistra.

QT interval dispersion (QTD) is considered to reflect the inhomogeneity of ventricular repolarization. Increased QTD has been shown to be positively correlated to complex ventricular arrhythmias (CVA) in many clinical conditions. The aim of this study was to assess QTD in arterial hypertension with left ventricular hypertrophy (LVH) and to verify the possible relationship between increased QTD and the presence of CVA in this population. We studied 100 patients with essential arterial hypertension, aged 25-78 years, mean 53. Half of the patients had LVH (group A); LVH was defined as diastolic septal thickness of 1.2 cm or more (by echocardiography). The septum was 1.1 cm or less in the patients without LVH (group B). QTc intervals were measured on the 12-lead surface ECG; QTD was defined as the difference between the longest and the shortest QTc. The presence of Lown class 2 (or more) ventricular arrhythmias on a 24-hour Holter monitoring was considered CVA. In the 100 patients studied, QTD ranged from 10 to 128 ms, mean 43.7 ms. It was longer in group A than in group B; 56.1 ms versus 51.5 ms (mean values). 14 patients in group A had CVA; they had a shorter QTD than patients without CVA: 51.2 ms versus 57.9 ms. In patients with essential arterial hypertension the presence of LVH is positively correlated to increased QTD. Increased QTD however does not seem to be an adjunctive risk factor for CVA in is population.

[Corrected transposition of the great vessels. A clinical review of 21 cases (author's transl)]. / Trasposizione corretta dei grossi vasi. Rassegna clinica e strumentale di 21 casi

Clinical and instrumental features of 21 cases of congenitally corrected transposition of the great vessels are reviewed. We confirm the rarity of the isolated form of the disease and its frequent association with ventricular septal defect and pulmonary stenosis. Electrocardiographically, the inversion of the first vectors is represented more frequently by the absence of the q wave on the left precordial leads than by the presence of the q wave on the right precordial leads; a very frequent electrocardiographic feature is a positive T wave on the right precordial leads. The angiographic image of the two ventricles in the lateral projection where they appear as crossing each other in their upper part were described. We confirm the poor results of the surgical treatment of the disease and the non-benign prognosis, because of the other associated cardiac anomalies.

Detection of specific protein binding to the SV40 early promoter in vivo.

The interactions in vivo between cellular proteins and the Simian Virus (SV40) early promoter region, contained in a plasmid capable of replicating in Cos cells, have been characterized by DNaseI and dimethyl sulfate (DMS) footprinting. The relative contribution of each GC-motif within the 21 bp repeat upstream element to transcription was first determined after transfection of Cos cells with either the wild type 21 bp repeats or recombinants where the GC-motifs were mutated either individually or in neighboring pairs. Mutation of GC-motifs III and VI was the most detrimental, mutation of GC-I, -IV and -V also decreased promoter activity but to a lesser extent, while mutation of GC-II had little effect on transcription. All six GC-motifs of the wild type 21 bp repeats were found protected from DNaseI nuclease attack in vivo though to varying degrees. Motifs GC-III, -V and -VI were more strongly protected than GC-I, -II and -IV. In vivo DNaseI footprinting of recombinants bearing mutations in the GC-motifs demonstrated the specificity of factor interaction and further indicated that, in agreement with the previously published in vitro results, the binding of factor(s) to each of the GC-motifs was independent. DMS protection experiments identified specific guanine (G) contacts characteristic of Sp1 binding to the GC-motifs and this in vivo pattern was compared to that obtained in vitro using a crude nuclear extract. These results indicate that the transcription factor Sp1 interacts in vivo with the GC-motifs of the SV40 early promoter.

Expression in Escherichia coli: purification and properties of the yeast general transcription factor TFIID.

A T7 RNA polymerase expression system has been used for the efficient expression of the yeast RNA polymerase general transcription factor TFIID (TFIIDY), the TATA-box factor (previously called BTF1) in Escherichia coli. Expression of the gene was performed at 25 degrees C instead of 37 degrees C to increase the total amount of soluble TFIIDY. Soluble TFIIDY was purified in three chromatographic steps and was eluted from the final column, a heparin-5PW HPLC column, in two peaks at 0.38 M (peak I) and 0.42 M (peak II) KCl in which this protein was 52% and greater than 95% pure, respectively. The protein in both peaks was active in an in vitro transcription assay. However, while TFIIDY from peak II was essentially indistinguishable from the material isolated from yeast, the protein of peak I differed in a number of biochemical characteristics, having a lower specific activity in an in vitro transcription assay and displaying an altered pattern of bands in a DNA band shift assay. Despite these differences, the proteins in both peaks have identical molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable N-terminal amino acid sequences, and apparently exist as monomers under the conditions used for the heparin-5PW chromatography.

[Left atrial enlargement. Electrocardiographic, radiographic and echocardiographic evaluation]. / Ingrandimento atriale sinistro. Valutazione elettrocardiografica, radiografica ed ecocardiografica.

Some ECG criteria and the radiologic method of Westcott are evaluated against echocardiography in the left atrial enlargement. ECG shows generally a very good sensitivity (72%); particularly when the diameter is more than 5 cm. The radiologic findings show significant correlative indexes in normal and in softly enlarged atria. In atria greater than or equal to 5 cm the method is not so precise; the reasons are discussed on an anatomical basis. We acknowledge an important place to the ECG described criteria and to the Westcott method in the depistage of the left atrium enlargement.

A yeast activity can substitute for the HeLa cell TATA box factor.

Most class B (II) promoter regions from higher eukaryotes contain the TATA box and upstream and enhancer elements. Both the upstream and enhancer elements and their cognate factors have regulatory functions, whereas the TATA sequence interacts with the TATA box factor BTF1 to position RNA polymerase B and its ancillary initiation factors (STF, BTF2 and BTF3) to direct the initiation of transcription approximately 30 base pairs downstream. In many respects, class B promoter regions from the unicellular eukaryote Saccharomyces cerevisiae are similarly organized, containing upstream activating sequences that bear many similarities to enhancers. Although they are essential for initiation, the yeast TATA sequences are located at variable distances and further from the start sites (40-120 base pairs), whose locations are primarily determined by an initiator element. The basic molecular mechanisms that control initiation of transcription are known to be conserved from yeast to man: the yeast transcriptional transactivator GAL4 can activate a minimal TATA box-containing promoter in human HeLa cells, and a human inducible enhancer factor, the oestrogen receptor, can activate a similar minimal promoter in yeast. This striking evolutionary conservation prompted us to look for the presence in yeast of an activity that could possibly substitute for the human TATA box factor. We report here the existence of such an activity in yeast extracts.

Class II (B) general transcription factor (TFIIB) that binds to the template-committed preinitiation complex is different from general transcription factor BTF3.

A class II (B) general transcription factor of 34 kDa has been purified from HeLa cells to apparent homogeneity. This factor appears to be transcription factor IIB (TFIIB), since it binds in vitro to template-committed preinitiation complexes formed between a template containing the TATA box/cap-site elements of the adenovirus type 2 major late promoter (Ad2MLP) and recombinant human or yeast TFIID (previously called BTF1) expressed in Escherichia coli. DNase I footprint studies show an extended pattern of protection of Ad2MLP TATA box/cap-site sequences when TFIIB is bound to template-committed complexes, even though TFIIB does not bind on its own to the template in the absence of TFIID. We also show that TFIIB is different from BTF3 by a number of criteria.

Addition of acetaldehyde to the N-terminus of a recombinant Schistosoma mansoni glutathione S-transferase upon high-level expression in Saccharomyces cerevisiae.

Intracellular expression of recombinant Schistosoma mansoni protein p28 (Smp28) in soluble form to a concentration of more than 6 g/l culture in Saccharomyces cerevisiae was accompanied by a post-translational modification, which occurred during the late stage of the culture. The modified protein, which had a reduced isoelectric point, was isolated by anion-exchange HPLC and characterized by tryptic mapping by means of on-line reversed-phase HPLC/electrospray mass spectrometry. Comparison with non-modified recombinant Smp28 allowed us to localize the modification to the N-terminal hexapeptide AGEHIK, which had an increased mass of 26 Da. Reversed-phase HPLC of the modified peptide with a shallow acetonitrile gradient revealed the presence of two components of identical mass and amino acid composition. Both peptides were inaccessible to N-terminal Edman sequencing, indicating that a rearrangement of the N-terminal region of recombinant Smp28 had taken place during tryptic digestion leading to two isomeric, N-terminally blocked peptides. Deuterium-exchange mass spectrometry showed that the modified peptides lacked two exchangeable protons, suggesting cyclic modifications implying the N-terminal amino group. Tandem mass spectrometry by means of the nano-electrospray technique and collision-induced dissociation allowed us to identify the modified sites as Ala1, His4 and Lys6 based on a characteristic modified a1 ion of Ala1 (70.0 Da), a modified immonium ion of His4 (136.0 Da) and a modified y1" ion (173.2 Da) of Lys6. Combination of all the above results led to the conclusion that recombinant Smp28 was initially modified at its N-terminus by addition of acetaldehyde to form an aldimine which rearranged during tryptic digestion to two different cyclic peptides.

Cloning of the gene encoding the yeast protein BTF1Y, which can substitute for the human TATA box-binding factor.

An activity (designated BTF1Y) in extracts of Saccharomyces cerevisiae can substitute for the human TATA box-binding factor BTF1 in a reconstituted transcription system containing the adenovirus 2 major late promoter, RNA polymerase B (II), and the basic transcription factors BTF2, BTF3, and STF. We have purified BTF1Y to homogeneity, using as assays reconstitution of in vitro transcription and DNase I footprinting on the TATA element. Both activities copurified with a 27-kDa polypeptide as determined by SDS/PAGE. Gel filtration indicated a molecular mass of 28 +/- 5 kDa under nondenaturing conditions, suggesting that the native BTF1Y protein is a monomer. BTF1Y was enzymatically cleaved, several peptides were sequenced, and appropriate oligonucleotide probes were synthesized to clone the BTF1Y gene from a yeast genomic library. The BTF1Y gene contains a 720-base-pair open reading frame encoding a protein of 27,003 Da. The recombinant protein expressed in HeLa cells exhibited the same chromatographic characteristics and in vitro transcriptional activity as BTF1Y prepared from yeast extracts, confirming the identity of the gene. Gene-disruption experiments indicated that the yeast BTF1Y gene is a single-copy essential gene.

TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III.

We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID-containing fractions stimulate U6 transcription in reactions containing rate-limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFIID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases.

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice.

Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein.