Characterization of Acinetobacter baumannii from intensive care units and home care patients in Palermo, Italy.

In this study 45 isolates of Acinetobacter baumannii identified from patients in intensive care units of three different hospitals and from pressure ulcers in home care patients in Palermo, Italy, during a 3-month period in 2010, were characterized. All isolates were resistant to at least three classes of antibiotics, but susceptible to colistin and tygecycline. Forty isolates were non-susceptible to carbapenems. Eighteen and two isolates, respectively, carried the bla(OXA-23-like) and the bla(OXA-58-like) genes. One strain carried the VIM-4 gene. Six major rep-PCR subtype clusters were defined, including isolates from different hospitals or home care patients. The sequence type/pulsed field gel electrophoresis group ST2/A included 33 isolates, and ST78/B the remaining 12. ST2 clone proved to be predominant, but a frequent involvement of the ST78 clone was evident.

Cerebral hemodynamic and metabolic changes in patients with fulminant hepatic failure during liver transplantation.

BACKGROUND: Cerebral autoregulation and metabolism may be seriously compromised in patients with fulminant hepatic failure (FHF). The mechanism responsible for the alteration in cerebral blood flow (CBF) has not been yet clearly defined; however, it is known that it does correlate with liver function. Orthotopic liver transplant (OLT) rapidly restores normal liver function, but little is known about the restoration of cerebral metabolism and hemodynamics. To investigate the relationship between liver function and CBF, we evaluated autoregulation and metabolic changes during OLT in six patients comatose due to FHF. METHODS: We evaluated autoregulation based on a linear regression analysis between mean arterial blood pressure and parallel CBF velocity (CBFV) changes using transcranial Doppler ultrasound. Cerebral metabolism rate was estimated by the arterial-jugular venous oxygen content difference (a-jDO2), while the percentile variation in cerebral metabolic rate for oxygen (CMRO2) was estimated using CBFV percentile variation rather than CBF percentile variation (eCMRO2). RESULTS: Prior to transplant autoregulation was impaired in all patients. However it markedly improved at the end of surgery (P <.05). The eCMRO2 improved as well, particularly among subjects who displayed prompt neurological recovery. In all patients the a-jDO2 was low before transplantation increasing to normal values at the end of surgery. CONCLUSIONS: A hallmark of FHF seems to be failure of autoregulation, which is linked to uncoupling between CBF and CMRO2 as attested by an a-jDO2 lower than normal in all patients (luxury perfusion). The recovery of liver function rapidly improves cerebral hemodynamics and metabolic stability. The study of autoregulation and eCMRO2 recovery using Doppler monitoring proffers the possibility to predict early graft function after liver reperfusion. In our patients eCMRO2 seemed to be associated with improved neurological outcomes.

Identification of Candida dubliniensis among oral yeast isolates from an Italian population of human immunodeficiency virus-infected (HIV+) subjects.

Candida dubliniensis, an emerging oral pathogen, phenotypically resembles Candida albicans so closely that it is easily misidentified as such. The aim of the present study was to evaluate the usefulness of two phenotypic methods, growth at 45 degrees C and 2,3,5-triphenyltetrazolium chloride (TTC) reduction, for confirming presumptive identification of C. dubliniensis and C. albicans by colony color on CHROMagar Candida (CAC) medium. A combination of these methods was used to establish the prevalence of oral C. dubliniensis in an Italian population of 45 human immunodeficiency virus (HIV)-infected subjects. Twenty-two samples (48.9%) were positive for yeasts on CAC medium producing a total of 37 fungal isolates. The colony color and 45 degrees C growth ability test correctly identified all C. dubliniensis and C. albicans isolates (5/37, 13.5%, and 16/37, 43.2%, respectively), while assessment of TTC reduction misidentified one C. albicans isolate. The isolation rate of C. dubliniensis was 11.1% (5/45 patients). All of the C. dubliniensis isolates were highly susceptible to fluconazole (MIC = 0.5 microg/ml). The combination of CAC medium screening with growth at 45 degrees C and TTC reduction tests may represent a simple, reliable and inexpensive identification protocol for C. dubliniensis.

Fluconazole susceptibility of Italian Candida dubliniensis clinical isolates determined by reference and simplified tests.

Candida dubliniensis ia an opportunistic pathogen mainly associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals. We recently recovered the first Italian clinical isolates of C. dubliniensis from the oral cavities of seven HIV-seropositive subjects. The in vitro susceptibility to fluconazole (FLCZ) of these isolates was determined according to the National Committee for Clinical Laboratory Standards (NCCLS) M27-A broth microdilution method for yeasts. All seven isolates of C. dubliniensis were susceptible to FLCZ (MICs < or =0.5 microg/ml). Results of this reference method were compared to those obtained with simplified tests, more adapted to routine evaluation in hospital laboratories. Fungitest and Sensititre YeastOne colorimetric microplate-based methods have been evaluated. The agar disk diffusion method has also been tested on two different media: RPMI 1640-2% glucose and High Resolution-2% glucose-0.5 microg/ml methylene blue. All of the simplified methods tested were able to correctly identify FLCZ-susceptibility of this group of Italian C. dubliniensis isolates.

Clinico-pathologic, immunohistochemical, and TUNEL study in early cardiac allograft failure.

Early cardiac allograft failure (ECAF) was defined as acute allograft failure in the early transplant period. The aim of this study is to elucidate the clinicopathological and immunohistochemical characteristics and the role of apoptosis in ECAF in nine patients. We reviewed preoperative clinical data and morphological data at the time of autopsy or retransplantation. We also performed TUNEL assay and immunohistochemistry to study fibronectin and tubulin beta-II. The average recipient and donor age was 48 +/- 10.3 and 28 +/- 7.11 respectively. Seven patients died at a mean time of 26 hours. The remaining two patients underwent retransplantation and are alive. The mean cold ischemic time was 124. 1 +/- 44.5 minutes. No patient had a panel reactive antibody >15% and lymphocytic crossmatch was positive in one case. All cases had grade 2-3 of coagulative necrosis, which correlated positively with fibonectin accumulation in myocyte cytoplasm, and cytoplasmic tubulin loss (p < 0.05). TUNEL technique showed in all cases some degree of DNA strand breaks in cardiomyocytes. Endothelium DNA strand breaks were seen in seven cases. Patients transplanted because of idiopathic dilated cardiomyopathy had a significantly higher degree of DNA strand breaks in cardiomyocytes and endothelial cells (p = 0.03 and p = 0.02) than those transplanted because of ischemic cardiomyopathy. These results indicate that ECAF may be caused by ischemic-reperfusion damage to the donor heart assessed by myocyte coagulative necrosis, fibronectin accumulation in myocytes, tubulin loss, and DNA strand breaks of cardiomyocytes and endothelium. The use of a combination of these techniques might be appropriate in the diagnosis of ECAF in endomyocardial biopsies when it is suspected clinically.

Tabulation of alcohol content of beer and malt beverages.

We present data from the analysis of the alcohol content of 391 beers and malt beverages available for sale in the State of Washington. The beverages were tested by gas chromatography for their alcohol content. Considerable variability in the alcoholic strength was found, even within the same class. Overall, the range of concentrations was 2.92% (v/v) to 15.66% (v/v). The alcohol content of beverages consumed is a critical factor in Widmark or volume-of-distribution-type calculations used to estimate blood or breath alcohol content from patterns of alcohol consumption. Using the correct alcohol content for beer, when the brand is known, can make a significant difference in the reliability of the calculation, and the data presented here should assist with optimizing the accuracy of the calculation.

Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves

The degradation of peroxisomal and nonperoxisomal proteins by endoproteases of purified peroxisomes from senescent pea (Pisum sativum L.) leaves has been investigated. In our experimental conditions, most peroxisomal proteins were endoproteolytically degraded. This cleavage was prevented, to some extent, by incubation with 2 mM phenylmethylsulfonylfluoride, an inhibitor of serine proteinases. The peroxisomal enzymes glycolate oxidase (EC 1.1.3.1), catalase (EC 1.11.1.6) and glucose-6-phosphate dehydrogenase (EC 1.1. 1.49) were susceptible to proteolytic degradation by peroxisomal endoproteases, whereas peroxisomal manganese superoxide dismutase (EC 1.15.1.1) was not. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from spinach and urease (EC 3.5. 1.5) from jack bean were strongly degraded in the presence of peroxisomal matrices. These results indicate that proteases from plant peroxisomes might play an important role in the turnover of peroxisomal proteins during senescence, as well as in the turnover of proteins located in other cell compartments during advanced stages of senescence. On the other hand, our data show that peroxisomal endoproteases could potentially carry out the partial proteolysis which results in the irreversible conversion of xanthine dehydrogenase into the superoxide-generating xanthine oxidase (EC 1. 1.3.22). This suggests a possible involvement of the peroxisomal endoproteases in a regulated modification of proteins.

Alcohol content of beer and malt beverages: forensic consideration.

Beer consumption is commonly an issue in a medico-legal setting, requiring estimates either of a likely blood alcohol concentration (BAC) for a given pattern of consumption or vice versa. Four hundred and four beers and malt beverages available for sale in the State of Washington were tested by gas chromatography for their alcohol content. Considerable variability in the alcoholic strength was found, even within the same class. Overall the range of concentrations was 2.92%v/v to 15.66%v/v. The mean alcohol concentration for ales was 5.51%v/v (SD 1.23%v/v), and for lagers, 5.32% (SD 1.43%v/v). Some specialty brews had characteristically higher or lower mean concentrations: ice beers 6.07%v/v, malt liquor 7.23%v/v, light beer 4.13%v/v, seasonal ales 6.30%v/v. Six brands of lager and four light beers account for the majority of all beer sales in the United States, and the mean alcohol concentration for these products was measured as 4.73%v/v and 4.10%v/v respectively. Few of the beers (17%) were labeled with respect to alcohol content, and in some cases, there was a significant disparity between the concentration listed on the label, and the measured alcohol concentration. Toxicologists need to exercise caution when performing Widmark type calculations, using all available information to select the most appropriate estimate for alcoholic strength of a beer or malt beverage.

Ethanol content of various foods and soft drinks and their potential for interference with a breath-alcohol test.

A variety of breads and soft drinks were tested and found to contain low concentrations of alcohol. The potential for these products to generate false readings on an evidential breath-alcohol instrument was evaluated. Alcohol-free subjects ingested these products and then provided breath samples into a DataMaster. It was found that breath samples provided immediately after consumption of some of these products, or with them still present in the mouth, did produce low levels of apparent breath alcohol, which may or may not be rejected as invalid by the breath-test instrument. If the subject swallowed or expectorated the food or beverage and then observed a 15-min deprivation period during which nothing was introduced into the mouth, the apparent effect was eliminated. These findings emphasize the need for the mandatory pretest alcohol-deprivation period and the benefits of duplicate breath sampling.

Lipid peroxidation, tissue necrosis, and metabolic and mechanical recovery of isolated reperfused rat heart as a function of increasing ischemia.

Isolated Langendorff-perfused rat hearts, after 30 min of preperfusion, were submitted to increasing times of global normothermic ischemia (1, 2, 5, 10, 20 and 30 min) or to the same times of ischemia followed by 30 min of reperfusion. Analysis of malondialdehyde, ascorbic acid, oxypurines, nucleosides, nicotinic coenzymes and high-energy phosphates was carried out by HPLC on neutralized perchloric acid extracts of freeze-clamped tissues. In addition, maximum rate of intraventricular pressure development and cardiac output of malondialdehyde, lactate dehydrogenase, oxypurines and nucleosides were monitored during both preperfusion and reperfusion. Besides decreasing energy metabolites and nicotinic coenzyme pool, prolonged ischemia produced oxidation of significant amounts of hypoxanthine and xanthine to uric acid and generation of detectable levels of malondialdehyde (0.002 micromol/g dry weight). After oxygen and substrate readmission, tissue and perfusate malondialdehyde increased only if previous ischemia was longer than 5 min, while lactate dehydrogenase was detected in perfusate of reperfused hearts following 10, 20, and 30 min of ischemia. Highest values of tissue malondialdehyde and total malondialdehyde output were recorded in reperfused hearts subjected to 30 min of ischemia (0.043 micromol/g dry weight and 0.069 micromol/30 min/g dry weight, respectively). Since tissue malondialdehyde was observed without detectable lactate dehydrogenase release in perfusate, it might be stated that malondialdehyde generation (i.e., lipid peroxidation) temporally preceded lactate dehydrogenase release (i.e., tissue necrosis). In reperfused hearts, evaluation of myocardial energy state and of mechanical recovery allowed us to determine times of ischemia beyond which reperfusion did not positively affect these metabolic and functional parameters. Main findings are that, under these experimental conditions, lipid peroxidation might be the cause and not the consequence of tissue necrosis and that duration of ischemia might be the factor deciding effectiveness of reperfusion.

A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes.

The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied. G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively. The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 microM, respectively. Dithiothreitol did not inhibit G6PDH activity. By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jiménez, Hernández, del Río and Sevilla (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes.

Evaluation of the effect of asthma inhalers and nasal decongestant sprays on a breath alcohol test.

The effects of eight prescription and non-prescription asthma inhalers and four over-the-counter nasal decongestants on the DataMaster, an evidential breath alcohol instrument, were evaluated. Subjects self administered the medication, and breath alcohol tests were administered immediately after use and following a 15 min waiting period. The only preparation which produced any effect on the instrument was Primatene Mist which contains 34% ethyl alcohol. The alcohol was, however, eliminated from the breath in the usual pattern of mouth alcohol elimination, and after 5 min there was no longer any effect. The inclusion of a 15 min deprivation period prior to an evidential breath test, during which time nothing is introduced into the mouth, is an adequate safeguard against interference with the test caused by alcohol containing inhalers.

Characterization of endoproteases from plant peroxisomes.

In this work, the characterization of endoprotease (EP) isoenzymes in peroxisomes is reported for the first time in cell organelles purified from pea leaves (Pisum sativum L.). A comparative analysis of the endo-proteolytic activity in peroxisomes purified from young (15-day-old) and senescent (50-day-old) leaves was carried out. Peroxisomes purified from senescent leaves showed a much higher endo-proteolytic activity than organelles from young plants. A 16 h incubation with exogenous substrates was the threshold time for the detection of a linear increase in the endo-proteolytic activity of peroxisomes from senescent leaves. Three EP isoenzymes (EP2, EP4 and EP5), having molecular masses of 88, 64 and 50 kDa respectively, were found in young plants by using SDS/polyacrylamide-gradient gels co-polymerized with gelatin. However, four additional isoenzymes (EP1, EP3, EP6 and EP7), with molecular masses of 220, 76, 46 and 34 kDa respectively, were detected in senescent plants. All the isoenzymes detected in peroxisomes from both young and senescent leaves were neutral proteases. By using different class-specific inhibitors, the electrophoretically separated EP isoenzymes were characterized as three serine-proteinases (EP1, EP3 and EP4), two cysteine-proteinases (EP2 and EP6) and a metallo-proteinase (EP7), and EP5 might be a metal-dependent serine-proteinase. Moreover, a peroxisomal polypeptide of 64 kDa was recognized by an antibody against a thiol-protease. The serine-proteinase isoenzymes (EP1, EP3 and EP4), which represent approx. 70% of the total EP activity of peroxisomes, showed a notable thermal stability, not being inhibited by incubation at 50 degrees C for 1 h.

Purification and properties of cytosolic copper, zinc superoxide dismutase from watermelon (Citrullus vulgaris Schrad.) cotyledons.

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3,450 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2-4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric point of 4.63 and 4.66.

Effects of increasing times of incomplete cerebral ischemia upon the energy state and lipid peroxidation in the rat.

Different times of incomplete cerebral ischemia (2, 4, 6, 8, 10 and 30 min) were induced by bilateral common carotid artery occlusion in anesthetized rats to evaluate the time course of changes in lipid peroxidation and energy metabolism. Analysis of malondialdehyde (used to assess the levels of lipid peroxidation), ascorbic acid, oxypurines, nucleosides, nicotinic coenzymes and high-energy phosphates, was carried out by high-performance liquid chromatography on neutralized perchloric acid extract of brain tissue. Under the present experimental conditions, malondialdehyde, nicotinic coenzymes and ATP catabolites (oxypurines and nucleosides) were affected by increasing times of ischemia, with respect to control sham-operated rats. In particular, the concentration of malondialdehyde, undetectable in control brains, increased from 1.26 nmol/g wet weight after 2 min of carotid clamping to 13.42 nmol/g wet weight at the end of 30 min of incomplete cerebral ischemia. The presence of oxidative stress was further supported by ascorbic acid depletion, which was particularly significant after 10 and 30 min of incomplete ischemia. Carotid clamping provoked an imbalance between energy production and consumption that was evidenced by a reduction in ATP and GTP concentrations and an increase in ATP degradation products such as AMP, oxypurines and nucleosides. A decrement in the sum of adenine nucleotides and the energy charge potential indicated a progressive malfunctioning of energy-producing metabolic cycles. A possible contribution to such a severe change in energy state might be related to depletion of NAD and NADP, particularly noticeable after the longest incomplete brain ischemia times, that should have provoked a consequent lessening of oxido-reductive reactions. Bilateral carotid clamping causes a significant reduction in brain oxygen and substrate supply that results in inhibition of energy metabolism and triggering of oxygen-radical-induced lipid peroxidation.

Presence and distribution of immunoreactive FMRFamide- and bovine pancreatic polypeptide-like peptides in a protochordate ascidian.

FMRFamide and bovine pancreatic polypeptide (BPP) immunoreactivities were detected in the cerebral ganglion and in the alimentary tract of the protochordate ascidian Styela plicata. Neurons expressing immunostain to FMRFamide were mainly found in the cortical zone of the ganglion and a network of immunoreactive beaded nerve fibers was present in the neuropil. Co-localization of FMRFamide- and BPP-like materials was demonstrated in few nerve cell bodies and both peptides likely play a neurotransmitter role. Parallel, endocrine-like cells containing FMRFamide-like materials were distributed among the esophageal and gastric epithelia, whereas no PP immunostaining was found. The tetrapeptide could be involved in the regulation of the digestive processes by means of endocrine and/or paracrine mechanisms.

Biological Consequences of Topography on Wave-swept Rocky Shores: I. Enhancement of External Fertilization.

Surge channels on wave-swept rocky shores are characterized by the violent hydrodynamic mixing that accompanies broken waves. It has been suggested that this mixing rapidly dilutes gametes shed into the surf zone, thereby severely reducing the fraction of eggs that can be fertilized externally. Although surge channels are well mixed within themselves, field experiments show that the exchange of water between these small embayments and the adjacent mainstream is surprisingly slow. Thus, surge channels may act as "containment vessels," limiting the rate at which gametes are diluted, and thereby enhancing the efficacy of external fertilization. Indeed, a mathematical model of fertilization in surge channels suggests that given a sufficient population of adult males within a surge channel, 80-100% of eggs may be fertilized. This result must be tempered, however, by the possibility that the small-scale shears induced by turbulence interfere with fertilization.

Efeito do Duovent (brometo de ipratrópio e fenoterol) na hiper-reatividade brônquica näo específica / Effect of Duovent (N-Isopropilatropin and fenoterol) in non-specific bronchial hyper-activity

As drogas beta-2-adrenérgicas e anticolinérgicas demonstraram uma açäo na modulaçäo da hiper-reatividade brônquica a vários estímulos (químico, físico, imunológico, farmacológico). O objetivo de nosso estudo é comparar a eficácia da combinaçäo do brometo de ipratrópio e fenoterol (Duovent) com a açäo individual das drogas, na prevençäo do broncoespasmo induzido por histamina. Vinte e seis indivíduos asmáticos atópicos foram examinados em um ensaio duplo-cego, durante o período assintomático, com um VEF, näo inferior a 20% do valor previsto normal. Durante 4 dias consecutivos todos os pacientes receberam 2 puffs (jatos), respectivamente, de Duovent (200 microng de fenoterol + 40 microng de brometo de ipratrópio), fenoterol (400 microng), brometo de ipratrópio (80 microng) e placebo, distribuídos ao acaso. As doses de histamina (PD20) necessárias para induzir o broncoespasmo foram avaliados após cada administraçäo da droga: após 2 h em 16 pacientes e após 5 h em outros 10 pacientes. Os dados foram transformados em valores log e a avaliaçäo estatística foi efetuada através da análise de variância com 2 variáveis. Este estudo mostrou que Duovent e fenoterol têm um efeito protetor contra o broncoespasmo induzido por histamina com um aumento significante nos valores do PD20 2 h (p < 0,01e 5 h (p < 0,05) após o tratamento em comparaçäo com o placebo. A inalaçäo de Duovent determinou um efeito mais protetor do que outras drogas, mas näo de forma significante quando comparado ao fenoterol. Algumas vantagens na modulaçäo da reatividade brônquica puderam ser observadas com o uso de Duovent, uma vez que com uma dose mais baixa da droga beta-2-adrenérgica pode-se obter os mesmos resultados sem efeitos colaterais