Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95% concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and ofXpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

Preserved xenogenic amniotic membrane as a patch on the repair of superficial corneal ulcers in rabbits

The aim of this study was to evaluate the effects of canine amniotic membrane, previously preserved in glycerin, used as a patch on the repair of experimentally-made superficial corneal ulcers and to compare corneal epithelization between the treated and non-treated groups. Xenogeneic amniotic membranes were collected aseptically and preserved in 99% glycerin at room temperature. Each animal was anesthetized and submitted to superficial corneal keratectomy of the left eye. The treated group received a fragment of canine amniotic membrane as a patch, while the control group had no treatment. The treated group showed blepharospasm, ocular discharge and conjunctival congestion. The membrane accelerated corneal repair in the beginning of the process, however, it delayed its conclusion (p<0.05). Treated eyes showed greater vessel formation and decreased corneal transparency (p<0.05). The stroma of the control group was thicker than that of the treated group (p<0.05). We suggest that amniotic membrane used in this manner can be applied as a therapy for superficial corneal ulcers in the beginning phases of the repair process.

Glucosamine and chondroitin sulfate in the repair of osteochondral defects in dogs - clinical-radiographic analysis

Among the proposed treatments to repair lesions of degenerative joint disease (DJD), chondroprotective nutraceuticals composed by glucosamine and chondroitin sulfate are a non-invasive theraphy with properties that favors the health of the cartilage. Although used in human, it is also available for veterinary use with administration in the form of nutritional supplement independent of prescription, since they have registry only in the Inspection Service, which does not require safety and efficacy testing. The lack of such tests to prove efficacy and safety of veterinary medicines required by the Ministry of Agriculture and the lack of scientific studies proving its benefits raises doubts about the efficiency of the concentrations of such active substances. In this context, the objective of this study was to evaluate the efficacy of a veterinary chondroprotective nutraceutical based on chondroitin sulfate and glucosamine in the repair of osteochondral defects in lateral femoral condyle of 48 dogs, through clinical and radiographic analysis. The animals were divided into treatment group (TG) and control group (CG), so that only the TG received the nutraceutical every 24 hours at the rate recommended by the manufacturer. The results of the four treatment times (15, 30, 60 and 90 days) showed that the chondroprotective nutraceutical, in the rate, formulation and administration at the times used, did not improve clinical signs and radiologically did not influence in the repair process of the defects, since the treated and control groups showed similar radiographic findings at the end of the treatments.

The Stoppa procedure in inguinal hernia repair: to drain or not to drain

OBJECTIVE: The objective of this study is to evaluate the benefits of drainage in the Stoppa procedure for inguinal repair. PATIENTS AND METHODS: The use of a suction drain was randomized at the end of the surgical intervention in 26 male patients undergoing inguinal hernia repair, divided into 2 groups: Group A, 12 patients undergoing drainage, and group B, 14 patients not undergoing drainage. On the second postoperative day, all patients underwent abdominal pelvic computed tomography scan examination to detect the presence of abdominal fluid collection. RESULTS: In group A, no patient developed fluid collection in the preperitoneal space, and 1 patient presented with an abscess in the preperitoneal space on the 15th postoperative day. In group B, 12 patients presented with fluid collections in the preperitoneal space on computed tomography scan evaluation. However, only 3 patients presented minor complications. None of the patients developed a major complication. CONCLUSION: The use of suction drainage with the Stoppa procedure does not provide any benefit.

Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin

OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.

Mitral valve repair. Quadrangular resection of the posterior leaflet in patients with myxomatous degeneration

OBJECTIVE - To analyze the immediate and late results of mitral valve repair with quadrangular resection of the posterior leaflet without the use of a prosthetic ring annuloplasty. METHODS - Using this technique, 118 patients with mitral valve prolapse who underwent mitral repair from January '84 through December '96 were studied. Age ranged from 30 to 86 (mean = 59.1±11.8) years and 62.7% were males. An associated surgery was performed in 22% of the patients, and coronary artery bypass graft was the most frequently performed surgery (15 patients - 12.7%). In 20 (16.9%) patients other associated techniques of mitral valve repair were used and shortening of elongated chordae tendineae was the most frequent one (6 patients). RESULTS - Immediate mortality was 0.9% (one patient). Long-term rates for thromboembolism, endocarditis, re-operation and death in the late postoperative period were 0.4%, 0.4%, 1.7% and 2.2% patients/year, respectively. The actuarial curve of survival was 83.8±8.6% over 12 years; survival free from re-operation was 91.8±4.3%, free from endocarditis was 99.2±0.8% and free from thromboembolism was 99.2±0.8%. In the late postoperative period, 93.8% of the patients were in functional class 1 (NYHA), with a complete follow-up in 89.7% of the patients. CONCLUSION - Patients with mitral valve prolapse who undergo mitral valve repair using this technique have a satisfactory prognosis over 12 years.

Mitral valve repair with a malleable bovine pericardium ring

OBJECTIVE: To describe a surgical procedure utilizing a malleable bovine pericardium ring in mitral valve repair and clinical and echodopplercadiographic results. METHODS: Thirty-two (25 female and 7 male) patients, aged between 9 and 66 (M=36.4±17.2) years, were studied over a 16-month period, with 100% follow-up. In 23 (72%) of the patients, the mitral approach was the only one applied; 9 patients underwent associated operations. The technique applied consisted of measuring the perimeter of the anterior leaflet and implanting, according to this measurement, a flexible bovine pericardium prosthesis for reinforcement and conformation of the posterior mitral annulus, reducing it to the perimeter of the anterior leaflet with adjustment of the valve apparatus. RESULTS: The patient survival ratio was 93.8%, with 2 (6.2%) fatal outcomes, one from unknown causes, the other due to left ventricular failure. Only one reoperation was performed. On echodopplercardiography, 88% of the patients had functional recovery of the mitral valve (50% without and 38% with mild insufficiency and no hemodynamic repercussions). Of four (12%) of the remaining patients, 6% had moderate and 6% had seigre insufficiency. Twenty-eight percent of class II patients and 72% of class III patients passed into classes I (65%), II (32%), and III (3%), according to NYHA classification criteria. CONCLUSION: Being flexible, the bovine pericardium ring fit perfectly into the valve annulus, taking into account its geometry and contractility. Valve repair was shown to be reproducible, demonstrating significant advantages during patient evolution, which did not require anticoagulation measures.

Remission of heart failure through endoluminal repair of femoral arteriovenous fistula with the use of a covered stent

We report the case of a 21-year-old male with high-output heart failure due to a femoral arteriovenous fistula caused by a firearm wound. A new balloon expandable stent covered with polytetrafluorethylene was implanted in the artery to occlude the arteriovenous fistula. The fistula was immediately occluded and the artery remained patent. On the following day, the patient felt much better, with no symptoms of heart failure. Additional follow-up is required to assure the usefulness of this less invasive procedure in the treatment of arteriovenous fistulas.

Surgical repair of a pseudoaneurysm of the ascending aorta after aortic valve replacement

We report the case of a patient with a pseudoaneurysm of the ascending aortic clinically diagnosed 5 months after surgical replacement of the aortic valve. Diagnosis was confirmed with the aid of two-dimensional echocardiography and helicoidal angiotomography. The corrective surgery, which consisted of a reinforced suture of the communication with the ascending aorta after opening and aspiration of the cavity of the pseudoaneurysm, was successfully performed through a complete sternotomy using extracorporeal circulation, femorofemoral cannulation, and moderate hypothermia, with no aortic clamping.

Repair of an Atherosclerotic Coronary Artery Aneurysm by Implantation of a Coronary Covered Stent

An atherosclerotic aneurysm of the right coronary artery complicated by a recent myocardial infarction was successfully treated with coronary artery stenting, using a device consisting of 2 stents with a layer of expandable polytetrafluorethylene (PTFE) placed between them. A follow-up angiograph 5 months after the procedure showed sustained initial results.