David operation: single center 10-year experience.

AIM: Aortic valve-sparing operation has been progressively widely performed for the treatment of aortic root aneurysm. Nowadays, this procedure has been proposed even in presence of a bicuspid aortic valve, severe aortic regurgitation or in primary aortic dissection repair. We present our ten-year experience focusing on mid-term echocardiographic follow-up. METHODS: Between June 2002 and February 2012, 139 patients (mean age of 61±12 years) underwent aortic valve-sparing operation with valve reimplantation. Twenty-seven patients (19%) had bicuspid aortic valve; in eighteen cases (13%) cusp motion or anatomical abnormalities concurred in determining aortic regurgitation and needed an adjunct cusp repair. A Gelweave Valsalva™ graft was implanted in all the patients. RESULTS: The mortality pre-discharge was 0.7% (1 patient). The cumulative 1-year, 5-years and 8-years survival rates were 99%, 93% and 87% respectively. Postoperative aortic regurgitation more than mild degree (>2+/4+) was the only significant risk factors for redo aortic valve surgery Freedom from reoperation due to aortic valve regurgitation was 96% at 1 year, 90% at 5 years and 86% at 8 years. When comparing freedom from reoperation in patients with bicuspid vs tricuspid aortic valve, no differences were found (P=0.31) and the rate of aortic valve reoperation was significantly higher (P<0.001) in patients who received leaflet's repair. CONCLUSION: The durability of valve reimplantation was found to be excellent in patients with tricuspid aortic valve and normal or nearly normal cusps. Cusp prolapse and complication after cusp repair turned out to be the main causes for early failure.

Port-access cardiac surgery: clinical experience with first 50 cases.

BACKGROUND: Minimally invasive cardiac surgery constitutes an expanding field for the treatment of many cardiac diseases. We analyze our experience with the first 50 patients operated upon using the port-access system. METHODS: From October 1999 to October 2000, 50 patients underwent cardiac operations with the port-access technique, for the correction of mitral and tricuspid disease, atrial septal defect closure, and removal of cardiac tumors. The mean age was 56.5 years. In 23 patients the mitral valve was repaired, in 14 cases a prosthetic replacement was performed, in 2 patients a paravalvular leak was resutured, 1 patient had a cardiac myxoma removed, and 1 patient had a correction of tricuspid regurgitation. In 9 cases an atrial septal defect closure was performed. RESULTS: One redo patient, with severe tricuspid regurgitation, needed conversion to sternotomy; in all the other cases the results of surgery were good and there were no surgical limitations. The mean cross-clamping time and bypass time were 53 +/- 19 and 77 +/- 27 min respectively. The mean in-hospital stay was 6 days. Re-exploration for bleeding was required in 7 patients (14%). Three patients (6%) presented with a postoperative neurological lesion; 2 recovered completely within 48 hours whereas 1 patient had permanent hemiplegia at discharge. There was one hospital death (an 81-year-old patient died of multiorgan failure on the thirteenth postoperative day). CONCLUSIONS: Minimally invasive port-access surgery is a reliable alternative to conventional surgery for the treatment of mitral and tricuspid disease, the removal of cardiac tumors, and the correction of atrial septal defects. It reduces trauma to the patient, provides a better cosmetic result and also allows for a faster recovery. Careful patient selection is important to avoid complications. Redo patients can be successfully treated and the risks of re-sternotomy avoided.

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II.

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged beta-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.

XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

[Myocardial revascularization in patients with severely depressed left ventricular function]. / La rivascolarizzazione miocardica nei pazienti con funzione ventricolare sinistra severamente depressa.

BACKGROUND: Improvements in anesthetic and surgical management of patients with left ventricular dysfunction have resulted in a decline in perioperative mortality and morbidity. Nevertheless, coronary artery bypass grafting (CABG) in patients with left ventricular ejection fraction < or = 0.30 remains a surgical challenge. METHODS: Fifty-one patients with end-stage coronary artery disease and left ventricular ejection fraction between 16 and 30% underwent CABG. Mean age at operation was 66.1 +/- 7.85 years. Selection criteria included the clinical diagnosis of ischemic heart disease with angiographic demonstration of critical coronary artery obstructive lesions. Mean number of grafts per patient was 2.94 (range 1-5). Average duration of cardiopulmonary bypass was 74.5 +/- 22.4 min and mean aortic cross clamp time was 47.6 +/- 17 min. RESULTS: No operative and in-hospital deaths occurred. Eight patients (15.7%) had postoperative low cardiac output syndrome, requiring intraaortic balloon counterpulsation. There were two major neurological complications (3.9%). There were four late deaths (7.8%), due to recurrence of untreatable congestive heart failure. Left ventricular ejection fraction increased from a mean of 25.51 +/- 4.75% preoperatively to 31.35 +/- 9.9% postoperatively (p < 0.001). Improvement in NYHA functional class (preoperatively 2.98 +/- 0.79 vs 2.35 +/- 0.6 postoperatively, p < 0.001) was found in this group at follow-up. CONCLUSIONS: CABG leads to an excellent prognosis in high risk patients with ischemic heart disease and low left ventricular ejection fraction, improving their functional and clinical outcome and consequently their life expectancy.

Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation.

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].

"Hybrid" and combined percutaneous and surgical intervention to treat selected cardiac patients: a new strategy.

The term "hybrid revascularization" describes the combined use of minimally-invasive surgery without cardiopulmonary bypass and percutaneous coronary revascularization in selected cases. The theoretical advantage of a less invasive surgical intervention must be weighted against the need for additional percutaneous procedures, with their own risks and limitations. We describe our initial experience with hybrid revascularization at the Istituto Clinico Humanitas in Milan. From 7/97 to 10/98, twelve patients underwent hybrid revascularization or a combined percutaneous and surgical intervention. A "classic" hybrid approach, consisting of minimally-invasive direct coronary artery bypass to the left anterior descending coronary artery and angioplasty or stenting of arteries in the right coronary artery or circumflex territories, was used in nine patients. In three patients, myocardial revascularization could be completed with percutaneous procedures after bypass surgery without cardiopulmonary bypass (in two patients because of severe aortic calcification) or valve surgery in a patient with two previous bypass operations. In-hospital complications were observed in three patients. Two required urgent median sternotomy (one for impending cardiac tamponade, one for conversion to bypass on extra-corporeal circulation). One patient developed atheroembolism after percutaneous intervention: after hospital discharge, there was a recurrence of symptoms, clinical deterioration with renal failure and eventually death. At a mean follow-up of 152 +/- 91 days (range 17 to 283) after minimally-invasive surgery and 166 +/- 122 days (range 13 to 397) after angioplasty, all surviving patients are well and free of anginal symptoms. Closer collaboration between surgical and interventional operators may offer a novel approach to effective treatment of difficult patient subsets. However, our initial experience suggests that a cautious evaluation of possible risks and benefits must carefully be entertained in each patient who may be considered a candidate for hybrid revascularization or combined percutaneous and surgical intervention.

Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein.

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex.

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Cockayne syndrome: defective repair of transcription?

In the past years, it has become increasingly evident that basal metabolic processes within the cell are intimately linked and influenced by one another. One such link that recently has attracted much attention is the close interplay between nucleotide excision DNA repair and transcription. This is illustrated both by the preferential repair of the transcribed strand of active genes (a phenomenon known as transcription-coupled repair, TCR) as well as by the distinct dual involvement of proteins in both processes. The mechanism of TCR in eukaryotes is still largely unknown. It was first discovered in mammals by the pioneering studies of Hanawalt and colleagues, and subsequently identified in yeast and Escherichia coli. In the latter case, one protein, the transcription repair-coupling factor, was found to accomplish this function in vitro, and a plausible model for its activity was proposed. While the E. coli model still functions as a paradigm for TCR in eukaryotes, recent observations prompt us to believe that the situation in eukaryotes is much more complex, involving dual functionality of multiple proteins.

Molecular characterisation of the human apo(a)-plasminogen gene family clustered on the telomeric region of chromosome 6 (6q26-27).

The genes coding for apo(a) and plasminogen belong to a family of related genes sharing several structural sequences like leader, kringle, and protease domains. YAC cloning has allowed to understand that all these genes are clustered within 400 Kb of genomic DNA on the telomeric region of chromosome 6 (6q26-27). We have now characterized the two remaining members of the apo(a) and plasminogen gene cluster. One of them was found to contain a leader highly homologous to that of apo(a) and plasminogen, followed by several kringle IV-like units, kringle V and protease domains although no tail sequences could be detected. This apo(a)-like gene was found to be expressed at the RNA level in liver although an in-frame stop codon was detected in one of its kringle units. The other member of the cluster besides the leader shows a plasminogen tail-like domain whose sequences contain a frameshift resulting in a stop codon; another mutation, destroying a consensus splicing site, has been found in a large intron separating the exon coding for the leader from the one encoding the tail-like sequences. The structural organisation of this cluster suggests that new arrangements of these four genes will be a likely finding.

[Reinterventions on cardiac valve prosthesis]. / Reinterventi su protesi valvolari cardiache.

From January 1978 to December 1984, 214 patients underwent a total of 243 reoperations for repair or replacement of a prosthetic heart valve. On the basis of the number of valve reoperations in the same anatomic position within the heart, the patients were divided into three groups. Overall hospital mortality was 23.4% (CL 20.3-26.7) in Group I (214 patients), 48% (CL 36.1-60.1) in Group II (25 patients), 25% (CL 3.3-62.6) in Group III (4 patients). Hospital mortality appeared to be related to urgency of reoperation (p less than 0.001 in Group I; p = 0.037 in Group II), primary indication for reoperation (p = 0.034 in Group I; p = 0.022 in Group II), association with other cardiac surgical procedures (p = 0.00253 in Group I). Hospital mortality in Group I was significantly higher (p = 0.0056) when reoperation was performed within one year after valve replacement. No significant differences in urgency and emergency rate were noted between reoperations on mechanical heart valves and bioprostheses. No significant differences in bleeding complications were noted between reoperations and initial valve replacement. Mean follow-up is 37.4 +/- 21.8 months (range 2 to 85 months). Actuarial survival rate is 82.8 +/- 3.1% at 1 year, 78.7 +/- 3.5% at 2 years and 71.5 +/- 5.1% at 5 years; 90.9% (CL 87.4-93.6) of the followed patients are in I or II NYHA class. The results appear to suggest that when significant (on clinical or instrumental criteria) prosthetic disfunction is diagnosed, reoperation should be undertaken early to minimize operative risk. The Authors point out that surgery in such patients also ensures satisfying long-term results.