Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris.

AIMS: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. METHODS AND RESULTS: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat /Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. CONCLUSIONS: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.

Auricular involvement of a multifocal non-AIDS Kaposi's sarcoma: a case report.

Kaposi's sarcoma (KS) is a multicentric, malignant neoplastic vascular disease, mainly involving skin and mucosae, characterised by the proliferation of endothelial cells. The aetiology of KS still is unknown. Nonetheless, it has been reported that several epidemiological and environmental factors may play a role in its pathogenesis. Viral factors (i.e. human herpes virus 8, HHV-8) have also been claimed to play a role in the onset of KS. Four main clinical presentations of KS have been described: classic (sporadic), African (endemic), iatrogenic (immunosuppression-associated) and AIDS-associated (epidemic). The authors present a case of KS involving the external ear of a HIVnegative patient with a history of non-Hodgkin lymphoma and tuberculosis.

Hearing threshold assessment in young children with electrocochleography (EcochG) and auditory brainstem responses (ABR): experience at the University Hospital of Ferrara.

OBJECTIVE: Electrophysiological evaluation is a fundamental procedure for the diagnostic assessment of hearing loss during infancy; in these cases, information concerning threshold level and auditory perception is particularly useful to establish a correct hearing rehabilitation program (hearing aids and cochlear implants). Purpose of this study is to underline the role of auditory brainstem responses (ABR) and electrocochleography (EcochG) in the definition of hearing loss in a selected group of children, referred to the Audiology Department of the University Hospital of Ferrara, for a tertiary level audiological assessment. METHODS: A retrospective study of the paediatric patient database at the Audiology Department of the University Hospital of Ferrara has been performed. In a period between January 2000 and December 2007, a total of 272 paediatric cases have been identified (544 ears). An EM 12 Mercury apparatus has been used for the electrophysiological threshold identification (ABR and EcochG). Recordings were carried out under general anaesthesia, in a protected enviroment. RESULTS: In 19 of the 272 paediatric cases selected--38 ears (7%), the results of threshold evaluation through ABR were uncertain. The Ecochg recording resulted crucial for the final diagnosis in terms of definition of the hearing threshold level, and it was then possible to ensure the better hearing rehabilitation strategy. CONCLUSIONS: ABR has to be considered the first choice in hearing assessment strategy, either for screening or for diagnosis in newborns as well as in non-collaborating children; ECochG still may be considered a reliable diagnostic tool.

Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country.

A sample of 22 Echinococcus granulosus isolates collected from 12 sheep and ten humans from a focus of cystic echinococcosis in western Turkey was examined by DNA sequencing of four mitochondrial genes (cox1, atp6, nad1, rrnS). Results demonstrated the presence of two species of E. granulosus complex, E. granulosus sensu stricto and E. canadensis. Of E. granulosus sensu stricto, the G1 genotype (including three microvariants) was found in 17 isolates from humans and sheep, the G3 genotype and an intermediate form G1/G3 in one isolate each (both from sheep). Of E. canadensis, the pig strain G7 was found in three isolates from sheep and human. This is the first report of this strain in Turkey. Its presence has implications for local control programs due to its shorter maturation rate in dogs compared with E. granulosus sensu stricto. Goat and/or wild boar are likely reservoirs for G7 in the region. We provided further data on the pattern and frequency of nucleotide substitutions within the G1/G3 cluster. Based on our results and GenBank records, G2 (Tasmanian sheep strain) is not considered as a discrete genotypic unit, as its sequences at polymorphic sites conform to microvariants of both G1 and (more often) G3.

Genetic variation within and between G1 and G3 genotypes of Echinococcus granulosus in Italy revealed by multilocus DNA sequencing.

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.

Proliferative peritoneal and pleural cestodiasis in a cat caused by metacestodes of Mesocestoides sp. Anatomohistopathological findings and genetic identification.

A 10-year-old female cat was brought to Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana for post-mortem examination. The animal used to live, together with 26 other cats, in the big terrace of an apartment at the 8th floor in Rome; and was always fed with industrial pet food. Anamnesis referred balance troubles, vomit and convulsions, during a couple of days, followed by sudden death. At necropsy, the cat presented mucoid rhinitis, purulent tracheitis, small areas of pneumonia, dark spots in the liver, catarrhal-hemorrhagic gastritis, fibrinous enteritis and meningeal hyperemia. Thoracic and abdominal cavities were completely invaded by hundreds of larval stages of cestodes. The same parasites were also included in nodules in pancreatic, lung and kidney parenchyma. Microscopic examination of parasites allowed their identification as larval stages (metacestodes) of cestodes of the genus Mesocestoides. The molecular genotyping of the metacestodes indicates a close relationship with members of the genus Mesocestoides, although a significant variation was found with respect to the available sequences of other species of the genus.

Occurrence of the G3 Indian buffalo strain of Echinococcus granulosus in cattle.

During a survey carried out to define the occurrence of Echinococcus granulosus in cattle bred in the province of Rieti (Central Italy), molecular diagnostics (PCR amplification and sequencing of a partial region of the mitochondrial CO1 gene) showed that 6/10 positive bovines harboured hydatid cysts (No.=16) genetically identical (95.8-100%) to the Indian buffalo genotype G3. As far the location of the 16 cysts, 11 of them were found in the lungs of three animals, whereas 5 cysts were in the liver of three parasitized hosts. The occurrence of genotype G3 in 60% of parasitized bovines living in an area never studied before provides more definite evidence about the existence of the strain in this region, and proves that cattle have to be considered a non-accidental host.

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing.

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.

Molecular identification of Anisakis species from Pleuronectiformes off the Portuguese coast.

Anisakid nematodes belonging to the Anisakis simplex complex are highly prevalent in several fish species off the coast of Portugal and are an important zoonotic problem in the Iberian Peninsula. Two reproductively isolated sibling species of the Anisakis simplex complex were identified from Pleuronectiformes inhabiting the Portuguese coast using restriction fragment length polymorphism (RFLP). Recombinant genotypes corresponding to presumptive Anisakis simplex sensu stricto and Anisakis pegreffii hybrids were also detected by this technique, as well as the species Anisakis typica. Although 25 species of Pleuronectiformes were investigated, Anisakis spp. larvae were only found in seven: Arnoglossus imperialis, Arnoglossus laterna, Lepidorhombus boscii, Citharus linguatula, Platichthys flesus, Dicologlossa cuneata and Solea senegalensis. The occurrence of hybrids in relatively sedentary fishes such as the Pleuronectiformes suggests that the Portuguese coast may constitute an area of hybridization and, therefore, is of particular interest for the study of the process of hybridization and speciation for these anisakids.

[Total arterial myocardial revascularization: surgical technique and mid-term results]. / La rivascolarizzazione miocardica arteriosa totale: tecnica chirurgica e risultati a medio termine.

The purpose of this study is to evaluate perioperative and mid-term results of total arterial coronary revascularization in our experience and to establish its safety. One-hundred and ninety-one patients underwent total arterial coronary revascularization at our Institution from February 1997 to August 2000. The mean age was 60.1 years. Of the patients, 45% had three coronary vessels disease, 12% a left main coronary artery disease and 2.6% of them were classified urgent. Mean ejection fraction was 59.6%. Three hundred and thirty-four comprehensive anastomoses were performed; mean grafts per patient was 1.8. A Y or T graft with the left internal thoracic artery was used in 21 patients. The in-hospital mortality rate was 1% (2 patients). Complications included low cardiac output syndrome in 2.8% of patients, acute myocardial infarction in 0.9% and acute renal insufficiency in 3.1%. Intraaortic balloon pump was used in 1.8% of patients. At a mean follow-up of 899 days the first 81 patients contacted are all free of angina but two (one experienced an acute myocardial infarction and another one died for a pulmonary neoplasia). We conclude that total arterial coronary revascularization is a safe surgical technique, providing excellent perioperative and mid-term results.

[Right ventricular function before and 6 months after aortocoronary bypass. A radioisotope angiographic study]. / La funzione ventricolare destra prima e sei mesi dopo bypass aorto-coronarico. Studio angiografico radioisotopico.

The aim of the present paper is the study of the changes induced on right ventricular function after myocardial revascularization with aortocoronary bypass graft. Two-three days before and 6 months after operation gated radionuclide ventriculography has been performed in 25 patients with previous myocardial infarction (MI), anterior in 16 patients and inferior in 9. At postoperative examination, right ventricular ejection fraction (EF) was slightly but significantly reduced (p less than 0.01), while peak ejection and filling rates were unchanged. Symmetric parameters of left ventricular function did not show changes at post-operative control, except a clear-cut rise of left ventricular peak filling rate (p less than 0.05). Before operation, in patients with previous anterior MI, left ventricular EF was slightly less and right ventricular EF slightly more than in patients with previous inferior MI; after operation right ventricular EF significantly decreased (p less than 0.01) only in patients with previous anterior MI. No correlation has been demonstrated in pre- postoperative changes of the observed parameters, neither between the two groups of previous MI nor between right and left ventricular cavity. Six months after myocardial revascularization, left ventricular performance, as examined with radionuclide angiography, was practically unchanged except for improvement of diastolic function, while right ventricular performance was moderately impaired, more in patients with previous anterior MI than in those with previous inferior MI.

[Left ventricular function a short time after myocardial revascularization surgery: radioisotope angiographic study]. / La funzione ventricolare sinistra a breve termine dall'intervento di rivascolarizzazione miocardica: studio angiografico radioisotopico.

The short-term effects after coronary bypass surgery on left ventricular function were studied in 65 patients with coronary artery disease. Each patient underwent gated radionuclide left ventriculography on the second-third day before and 5-7 days after coronary bypass surgery. After surgery, global ventricular ejection fraction had a significant increase, anteroseptal wall motion was unchanged, while apical motion and mainly inferoposterior motion were significantly increased. The most important improvement of inferoposterior motion was observed in 18 patients who had complete revascularization of the previously infarcted inferior segments, while in the 11 patients with incomplete revascularization of the same area, inferoposterior motion had a slight increase. There was a slightly significant direct linear correlation between size and direction of the anteroseptal and inferoposterior motion changes. The patients were divided in subgroups related to the site and number of obstructed coronary vessels as well as of implanted grafts; the computed parameters showed similar changes in all subgroups. After surgery, peak ejection rate showed an evident increase, and time to peak reciprocally shortened. Peak filling rate, which had reduced preoperative values, was found clearly improved, with a corresponding shortening of the time to peak; these results confirmed that diastolic ventricular damage was a reversible disturbance related to myocardial ischemia. In 8 of the 65 patients, 24 hours urinary catecholamine levels showed similar values at both pre and postoperative radionuclide controls. In conclusion, the radionuclide angiography showed an evident improvement of left ventricular function during a short period following surgery, suggesting a better myocardial perfusion after revascularization.(ABSTRACT TRUNCATED AT 250 WORDS)

[Analysis using scintigraphy with thallium-201 of the perfusion picture before and after myocardial revascularization]. / Analisi mediante scintigrafia con tallio-201 del quadro perfusionale prima e dopo rivascolarizzazione miocardica.

The present study was undertaken to investigate the effects of coronary artery bypass surgery on myocardial perfusion analyzed by thallium-201 scintigraphy. In 34 patients early thallium-201 imaging, repeated 4 hours later at rest, was performed 2-3 days before and within 1 year following myocardial revascularization. Before surgery, exercise ECGs were positive in 22/34 patients, with scintigraphic perfusion defects in 33/34 patients; thallium-201 imaging showed reversible in 77 and irreversible perfusion defects in 51 myocardial segments. After surgery, exercise ECGs were positive in 3/34 patients, while thallium-201 perfusion data improved in 27, were unchanged in 4 and worsened in 3 patients. After surgery, thallium-201 imaging showed a remarkable improvement, with decreased number of reversible (8 segments) and irreversible (32 segments) perfusion defects. Ten of the 22 patients with previous myocardial infarction had improved irreversible perfusion defects in myocardial segments that were infarcted. Qualitative thallium-201 scintigraphy showed that myocardial revascularization exerted a clear favorable influence on reversible perfusion defects and in part on irreversible perfusion defects, suggesting that persistent defects are compatible not only with scar tissue, but also may represent hypoperfusion of viable myocardium.

[Surgical treatment of infective endocarditis]. / Il trattamento chirurgico dell'endocardite infettiva.

Since 1975 to 1979 24 patients affected by acute infective endocarditis were operated of valvular replacement. All patients presented increasing cardiac failure unresponsive to medical treatment; 48% of the cases had positive blood culture. In 5 patients the infective endocarditis occurred on previously implanted prosthetic valve. Surgical findings consisted in rupture of chordae tendineae, vegetation ad perforation of valvular cusps. Prosthetic valvular replacement was performed in all cases using mechanical valve (22) or porcine heterograft 85). Surgical mortality was 8,3%, while late mortality occurred on 12.5% of cases. The diagnostic considerations, concerning this disease, the surgical results and long term management are discussed.

[Water-electrolyte and acid-base changes during extracorporeal circulation]. / Variazioni idroelettrolitiche ed acido-basiche durante circolazione extracorporea.

Changes in fluid, electrolyte and acid-base balance were evaluated in 8 patients during extracorporeal circulation. H2O increase and osmolarity and colloid osmotic pressure decrease, due to priming and perfusion technique used for cardiopulmonary by pass, were found. Mild respiratory alkalosis was also present. K+ serum, because of KCL supplements, was always maintained within the normal values, while Na+ serum concentration was low, despite of large amounts of Na+ infused during perfusion. After examination of disturbances in fluid, electrolyte and acid-base balance, the Authors report the performed corrections and emphasize the importance of a prompt recognition and the immediate therapy of these changes to avert serious cardiac impairments during extracorporeal circulation or during the immediate postoperative period.

[Is aortocoronary bypass indicated in patients with lesions of the common trunk of the left coronary artery incapable of complete revascularization?]. / E' indicato sottoporre a by-pass aorto-coronarico pazienti con lesioni del tronco comune della coronaria sinistra suscettibili di rivascolarizzazione incompleta?

Obstructive lesions of the left common coronary trunk need surgery if our operability criteria: possibility of at least making a by-pass on the left descending anterior coronary branch and a not seriously compromised miocardial contractility exist. Of the 25 patients operated, there was a mortality of 8% and 3 intra-and post-operatory infarcts; two other patients died after some time (one for extracardiac causes). Other infarcts were not observed. 48% of the patients had incomplete riva-scolarization which after some time had an effect on the objective state of the patients but not on their survival. The authors conclude their studies by affirming that total surgical correction is indicated even on non susceptible patients on the basis of coronarography.

[Effect of cardiopulmonary bypass on serum digoxin concentration (author's transl)]. / Influenza della circolazione extracorporea sul tasso ematico della digitale: osservazioni preliminari

The use of digitalis following cardiopulmonary bypass is studied by digitalemia's radioimmunoassay. The Authors does not find relation between digitalis and post operative arrhytmias. They think digitalis's tolerance is very good but suggest that further studies are necessary to state the correct use of the drug after open heart surgery.