We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.

Chromosome Deletion↗

PubMed1985

[Sarcoma of the heart. Apropos of 2 cases].

Sarcoma of the heart is a very rare condition. We present here two cases with very different clinical expression. The first patient, a 15-year old girl, presented with extremely severe ventricular arrhythmia, whereas the second patient, a 63-year old woman, mainly had signs of congestive cardiac failure. A diagnosis of cardiac tumour was made in the first case on the basis of ultrasonographic results which clearly showed the tumour developed from the walls of the left ventricle. In the second case, the tumour involved the right ventricular wall, the deformation of which was undetected by ultrasounds and only suspected at angiography. Both patients died rapidly, and the diagnosis of sarcoma was confirmed at post-mortem examination.

Adolescent↗

PubMed1985

Leptospiral exposure in Detroit rodent control workers.

Antibody levels to L. icterohemorrhagiae of rodent control workers and two control groups were compared with antibodies against five other leptospiral serotypes carried predominantly by species other than rats. Rodent control workers contact urban rats, 77 per cent of which are carriers of leptospiral organisms. Rodent control workers had higher antibody levels against L. icterohemorrhagiae than control groups (OR 4.37, 99 per cent CI = 3.0, 6.3 and OR 11.08, 99 per cent CI = 5.6, 22). Antibodies against the other five serotypes were almost non-existent in the study group.

Adult↗

PubMed1985

Cardiac arrhythmias treated by catheter ablation methods.

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Arrhythmias, Cardiac↗

PubMed1985

[Effect of oral propafenon on accessory pathways of atrioventricular conduction].

Fifteen consecutive patients with accessory AV pathways comprising 12 cases of the Wolff-Parkinson-White syndrome and 3 cases with exclusively retrograde conduction diagnosed by endocavitary electrophysiological investigation were studied. 5 patients had paroxysmal atrial fibrillation and the other 10 had reciprocating orthodromic tachycardia. 12 patients underwent electrophysiological investigation to measure the refractory periods and anterograde and retrograde conduction times in sinus rhythm and induced reciprocating tachycardia treatment with oral propafenone at a dose of 900 mg/day for 3 to 6 days. The refractory periods of anterograde conduction and the conduction times were then measured using an endocavitary atrial recording electrode left in position after the initial investigation. The anterograde Wenckebach point decreased from 259 +/- 35/min to 158 +/- 13/min (p less than 0.001). The refractory periods were increased from 270 +/- 45 ms to 371 +/- 100 ms (p less than 0.05). Atrial fibrillation could only be initiated in 2 cases with a mean rate which had decreased from 258 to 130/min. Reciprocating tachycardia could only be initiated in 5 cases and then with a slower rate (average cycle length increasing from 352 +/- 52 ms to 430 +/- 41 ms). Eight of these patients continued the treatment for over one year without recurrence of their arrhythmias, confirming the long-term efficacy of propafenone in the prevention of arrhythmias complicating accessory AV conduction pathways.

Administration, Oral↗

PubMed1985

Treatment of the perineum by pulsed electro magnetic therapy.

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Diathermy↗

PubMed1985

[Angiocardiographic diagnosis in right ventricular dysplasias].

Right ventricular dysplasia (RVD) is characterised by fatty and fibrous infiltration of the right ventricular wall and usually presents clinically with ventricular arrhythmias. The aim of this study was to determine the morphological changes of RVD and establish diagnostic criteria applicable in cases with atypical clinical signs or electrocardiographic changes. The angiocardiographic data of 10 cases of RVD was reviewed. 8 cases were complicated by right ventricular arrhythmias. The other 2 cases were not arrhythmogenic but had highly suggestive ECG changes; there were no other causes of right ventricular disease. Selective right ventriculography was performed with the catheter positioned in the inflow tract distal to the moderator band. In the 8 cases without tricuspid regurgitation there was one constant finding: the presence of parietal fissuration with massive impregnation of the ventricle distal to the moderator band. Two signs were commonly observed; the stagnation of contrast in the inferior regions of the right ventricle during left ventricular opacification (6/8 cases); irregular opacification of the pulmonary infundibulum (5/8 cases); an infundibulor aneurysm was observed in 1 case. Biometrical data was normal in all 8 cases compared with a control group of 10 normal subjects: the right ventricular disease was segmental and not diffuse. It was associated with a moderate alteration of left ventricular function and with segmental abnormalities of wall motion in 2 cases. None of these signs were observed in the right ventriculographies of 7 cases of typical dilated cardiom-opathies. These 4 signs were not found in 2 cases with tricuspid regurgitation where changes were essentially limited to a dilatation of the right ventricular inflow tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1985

[Emergency treatment of chronic ventricular tachycardia after myocardial infarction by endocavitary fulguration].

Endocavitary catheter ablation consists of delivering an impulse of 160 to 240 joules via a catheter used for electrophysiological investigation resulting in an electrical discharge which, in addition to its thermal effects may alter the arrhythmogenic substrate mechanically. This method was used in 2 patients with resistant and recurrent VT after myocardial infarction complicated by ventricular aneurysm. Two sessions of catheter ablation were necessary in both patients, but in the second one a single shock was successful in critically ill patients with VT. The first patient has been followed up for 20 months and the second for 13 months. Under prophylactic antiarrhythmic therapy, neither patient has had recurrence of the ventricular arrhythmias which had previously led to their hospitalisation. Catheter ablation is therefore presented as a technique which may be performed in the electro-physiological laboratory and repeated in cases of incomplete efficacy. This method may be used successfully in the treatment of chronic VT after myocardial infarction complicated by ventricular aneurysm.

Aged↗

PubMed1985

WAIS-R factor structure in psychiatric and general medical patients.

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Adult↗

PubMed1984

Phosphoenolpyruvate-dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system.

Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P-IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIglc of S. typhimurium. Here we present 1H-NMR measurements of IIIglc and P-IIIglc from E. coli which further substantiate that the phosphoryl group in P-IIIglc is linked to the N-3 position of a histidyl residue. After phosphorylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate-dependent phosphorylation.

Binding Sites↗

PubMed1984

The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase.

The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.

Adenylate Kinase↗

PubMed1984

Mitochondrial adenylate kinase (AK2) from bovine heart. Homology with the cytosolic isoenzyme in the catalytic region.

The adenylate kinase isoenzyme located in the intermembrane space of mitochondria, AK2, is a monomeric protein of Mr 30000 which catalyzes the reaction ATP + AMP + AMP in equilibrium 2 ADP. The protein was reduced and S-carboxymethylated with iodo[14C2]acetate. Using a Laursen sequenator, the N-terminal sequence of S-carboxymethylated AK2 was determined as Ala-Pro-Asn-; in some batches of the isolated protein the N-terminal dipeptide portion was missing. The C-terminus of AK2 was found to be Met. Cleavage with CNBr yielded eight fragments which could be isolated in one step using high-performance size-exclusion chromatography. They ranged in size over 4-88 amino acid residues, the total being approximately 270 residues. All CNBr fragments were overlapped with Met-containing tryptic peptides of AK2. The N-terminal 111 residues of AK2 were sequenced. Except for an N-terminal extension of nine residues, this segment of AK2 could be aligned with the sequence 1-104 of cytosolic AK1. Allowing for two deletions in AK2, 43 of the 102 aligned residues are identical. Since this section contains the catalytic residues such as His-36 and Asp-93, we conclude that AK1 can serve as a three-dimensional model of AK2 in mechanistic and drug-designing studies. preliminary sequence results on AK2 beyond position 104 show that AK2 here contains a wing of approximately 50 residues which has no counterpart in AK1. The chain folds of the adenylate kinase isoenzymes are similar again from a position corresponding to residue 115 of AK1 onwards. The additional structural motifs of AK2 are probably related to the location of this isoenzyme in the mitochondrion.

Adenylate Kinase↗

PubMed1984

The 5'-terminal sequence of U1 RNA complementary to the consensus 5' splice site of hnRNA is single-stranded in intact U1 snRNP particles.

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.

Autoantibodies↗

PubMed1984

Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro.

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible lambda pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to 10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(-) orientation is as efficient as the switch from G(-) to G(+).

Journal Article↗