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Background: In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA., Results: In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the M. smegmatis region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of M. tuberculosis and M. smegmatis were inserted in a plasmid between the sigA promoter and the lacZ reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the katG translation start codon, increased beta-galactosidase activity and stabilized the lacZ transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of M. smegmatis was followed by an increasing number of katG codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the katG transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing., Conclusion: This is the first reported case in mycobacteria, in which both a polypurine sequence and translation initiation are shown to contribute to mRNA stability. The furA-katG mRNA is transcribed from the furA promoter and immediately processed; this processing is prevented by a double stranded RNA at the cutting site, suggesting that the endoribonuclease responsible for the cleavage cuts single stranded RNA.

In the Escherichia coli satellite phage P4, transcription starting from PLE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t151 site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at PLE and PLL promoters, respectively, and terminating at the intracistronic t151 site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and alpha genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t151 transcription termination site within the 7633-7361 region between orf151 and gene cnr. A potential stem-loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414-7385.

The genetic element P4 propagates in its host Escherichia coli both as a satellite phage and as a plasmid. Two partially overlapping replicons coexist, namely replicon I and replicon II. The former is composed of two sites, ori1 and crr, and depends on P4 alpha gene product for replication. The P4 alpha protein has primase and helicase activities, and binds specifically to both ori1 and crr. Replicon II is composed of two sites, ori2 and crr, and its replication also depends on P4 alpha primase and helicase activities. In replicon II, the alpha protein binds only crr. Here we show that for replicon II the relative orientation of ori2 and crr is essential for replication to occur. Furthermore we delimit ori2 to a 22 bp region (6234-6255), internal to the alpha gene, sufficient for replicon II replication. We mutagenized this region and identified two mutants, which carry one and two base substitutions, respectively, that prevent replicon II replication. In electrophoretic mobility shift experiments of ori2, ori1, and crr DNA fragments with E. coli extracts, ori2 was not shifted, whereas both ori1 and crr were specifically bound, suggesting that other host protein(s), beside P4 alpha, are able to bind to these cis essential regions. Apparently, no binding to ori2 could be identified, thus suggesting that neither alpha nor other bacterial proteins specifically bind to this region.

The use of a patch after carotid endarterectomy (CE) is recommended to reduce the incidence of restenosis. Most studies on this subject report the implantation of saphenous vein or PTFE patches, because polyester has always been considered to be a thrombogenic material. The purpose of this study was to evaluate the thrombogenicity of a knitted polyester patch passivated by fluoropolymer surface treatment (FPD patch), which experimental studies have demonstrated to be less thrombogenic than other materials. This prospective, randomized study was performed in 22 patients who underwent CE. In 11 patients the arteriotomy was sutured directly, while in the other 11 an FPD patch was applied. Patients' 111in-oxine labeled platelets were reinjected on the first postoperative day, and scintigraphies were performed after 4, 24, and 48 hr, respectively. The study confirmed that an FPD patch is no more thrombogenic than a simple carotid endarterectomy. The application of the patch, therefore, can be recommended to reduce restenosis without any adjunctive thromboembolic risk.

The ischemia-reperfusion syndrome, first described by Haimovici in 1960, is a severe complication following surgery for acute ischemia. We evaluated the incidence of this complication in 264 patients operated on between 1972 and 1981 (1st group) and compared it with another of 392 patients operated on between 1982 and 1991 (2nd group), our aim being to assess the effects of pharmacological prophyiaxis based on preoperative overhydration followed by an intra-arterial bolus of 250 ml 14/1000 HCO3-, containing 1 g dexamethasone and 2500 I.U. sodium heparin, injected into the femoral artery before suturing the arteriotomy. This regimen was based on the measurement of myoglobin and glutathione levels respectively carried out in two subgroups of 25 patients. The results of experimental ischemia-reperfusion syndromes induced in animal using radical scavengers and membrane-protective compounds were also taken into consideration. Following experimental research on sheep, 5 patients in the second group with very severe ischemia due to aortic occlusion received local dialysis in the extracorporeal circulation using hemodialysis or hemofiltration techniques. Mortality was 6.3% in the first group and 5.4% in the second, while the amputation rate was 3% and 1.8% respectively. The overall incidence of the reperfusion syndrome was 3% in the 1st period and 1.8% in the second. Our findings confirm the protective effect of hyper-hydration, radical scavengers and dexamethasone in the ischemia reperfusion syndrome, and indicate that local hemodialysis is a useful adjunct in very severe ischemia.

Surgical treatment for graft thrombosis is often difficult and its outcome is complicated by a high incidence of rethrombosis. The records of 803 patients (1261 limbs) treated with aortofemoral bypass graft for peripheral vascular disease were reviewed. Among these, 71 patients (63 men and eight women with a mean age of 60.2 years) needed an overall number of 125 reoperations. The mean follow-up was 6.3 years. Smoking was significantly higher in these patients compared with those who were not reoperated on. Intimal hyperplasia was the most common lesion responsible for graft thrombosis. Before 'redo' surgery 20 patients with recent thrombosis received thrombolytic treatment; one was subsequently treated with percutaneous transluminal angioplasty and the others underwent reoperation. The best patency rates were achieved with reconstruction of the distal anastomosis with a graft interposition or with a patch angioplasty. Isolated thrombectomy was complicated by early rethrombosis in 53.3% of cases. The overall amputation rate was 14%, the operative mortality rate 5.6%, and the incidence of complications 16.8%. At follow-up a cumulative secondary patency rate of 81.7% at 5 years (74.5% at 10 years), a limb salvage rate of 80.5%, and a survival rate of 89.2% at 10 years were recorded. It is concluded that the only indications for redo surgery following graft thrombosis are severe limb ischaemia or disabling claudication.

Hypoplasia of the distal abdominal aorta, first described by Quain in 1847, is rarely associated with atherosclerotic occlusive disease. The condition usually occurs early in life; the principal clinical features are hypertension and arterial insufficiency of the lower extremities which is often well tolerated. Some 20 patients had operations for abdominal aortic hypoplasia; 16 underwent prosthetic reconstruction and four had aortoiliac endarterectomy with angioplasty. Reconstructive techniques were influenced by narrow renal arteries and by the presence of extensive atheroma. The long-term prognosis is often poor, especially in patients with extensive disease.

The results of carotid endarterectomy in 521 patients were analyzed to verify whether the early and late outcome in young patients is worse than in older patients, such as the results observed in peripheral arterial reconstruction in young adults. The patients were subdivided into 3 groups: 105 under 55 years, 397 aged between 56 and 74 years and 19 over 75 years. Young patients presented less risk factors and associated diseases than older ones, with the exception of smoking-habit; on the other hand, they presented a higher incidence of early post-operative thrombosis, myocardial infarction and of recurrent stenosis at follow-up. This trend has already been observed in other series. Young age and smoking habit have also been significantly related with recurrent carotid stenosis. In spite of these complications, carotid endarterectomy is indicated in severe stenosis or complicated carotid plaques, in young adults, because of a low operative mortality and a better trend in the follow-up period; however, the pre-operative evaluation needs a more accurate study and, when possible, the normalization of metabolic risk factors and the elimination of smoking.