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.

Hyperglycemia and prognosis of acute myocardial infarction in patients without diabetes mellitus.

The present study assessed the prognostic value of hyperglycemia--a common feature in the early phase of acute myocardial infarction (AMI)--in 330 nondiabetic patients. Seventy-nine known diabetics and 10 (3%) unknown diabetics--diagnosed before discharge by stable glycosylated hemoglobin greater than 6.9% and by oral glucose tolerance testing--were excluded. Thirty-three (10%) patients died. The mortality rate was higher in women, in patients with anterior AMI, in older patients (greater than 65 years) and in the presence of heart failure. It was highest in patients with cardiogenic shock (24/36 vs 9/294; p less than 0.0001). Admission plasma glucose was significantly higher in nonsurvivors than in survivors (163 +/- 60 vs 114 +/- 36 mg/dl; p less than 0.0001). Mortality rate increased with increasing admission plasma glucose: 3% in normoglycemic patients (less than or equal to 120 mg/dl) versus 15% in patients with borderline plasma glucose (121 to 180 mg/dl) versus 43% in hyperglycemic patients (greater than 180 mg/dl) (p less than 0.0001). Multiple regression (stepwise) analysis identified cardiogenic shock, infarct site and age as the major determinants of mortality, while admission plasma glucose failed to reach full statistical significance (p = 0.067). Hyperglycemia was related to all 3 of these independent prognostic factors; when age and infarct site were accounted for, hyperglycemia was significantly associated with heart failure only and this association was characterized by a remarkable mortality rate. In nondiabetic patients with AMI, hyperglycemia is a correlate of heart failure and, therefore, an important factor of prognosis.

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.

Effects of esmolol on airway function in patients with asthma.

In a double-blind, randomized, crossover study in ten patients with asthma, the effects on specific airway resistance of esmolol, a new ultra-short-acting beta 1-selective adrenoceptor blocker, were compared with those of placebo. Specific airway resistance was measured during increasing doses of esmolol infusion, during dry air provocation tests, and following isoproterenol inhalation. These same studies were later carried out on six of ten patients following intravenous propranolol infusion. All patients were able to tolerate the maximum dose of esmolol (300 micrograms/kg/min); treatment differences between esmolol and placebo were not found. In contrast, intravenous propranolol produced marked symptomatic bronchoconstriction after the lowest dose (1 mg) in two of six patients. Esmolol produced slight but statistically significant enhancement of patients' sensitivity to dry air provocation. Similarly, a slight but significant inhibition of bronchomotor sensitivity to isoproterenol was noted during esmolol infusion. After infusion of 5 mg of intravenous propranolol, one of four patients had a clinically significant increase in sensitivity to dry air. It is concluded that esmolol, because of its short duration of action and relative lack of effect on airway resistance, may be preferred over propranolol in patients with asthma who require treatment with an intravenous beta-blocking agent.

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.

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.

Profile of the hardy NICU nurse.

Interviews with our long-term NICU nurses correlated with Kobasa's hardiness research. our nurses see themselves as in control of their patients, who are entrusted to the nurses by the parents. They are committed to their work, which they view as essential. They are constantly challenged by the technological improvements and demands of their work. And, adding a fourth personality characteristic to Kobasa's work, they see companions as an integral part of their ability to cope with stress. We are interested in further investigating the companionship component and its importance for hardy NICU nurses, as well as the effects of the other hardiness characteristics. Our initial findings in our interviews, as well as further research in this area, may help in evaluating current unit policies and procedures and how they support these coping mechanisms. Our next step is to formally study our nurses and their hardy-to-stress personalities to determine whether we can effectively use a hardiness scale to direct nurses to high- or low-stress units. Another interesting question is whether nurses can be taught to become hardy. Much previous research on stress, burnout, and the nursing profession has focused on external factors and their effects on nurse retention. Utilizing Kobasa's and others' work on hardiness, we hope to learn more about the introspective factors and their relationship to the success of long-term NICU nurses.

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.

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.

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.

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.

Evaluation of the activity of the carbocysteine-sobrerol combination on mucus spinnability.

Among the rheological properties of bronchial mucus, "spinnability", i.e. the ability to form threads under the effect of traction, should be regarded as the most closely related to the mucociliary transport function. In the present study the "spinnability" parameter was included in the functional tests aimed at evaluating the efficacy of the carbocysteine-sobrerol combination in 16 patients suffering from chronic abstructive lung disease with bronchial hypersection. Treatment was administered for ten days under double-blind conditions compared with a placebo. The results obtained showed the tested combination to be able to favourably affect all the most important rheological parameters of mucus, including spinnability, leading to a rapid disappearance of signs and symptoms and to the improvement of the most important respiratory function indexes.

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.

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.

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.

Influence of asthmatic and rhinitic symptomatology duration on bronchial responsiveness to histamine.

Our Study aimed to investigate the influence of the time in years elapsed from the onset of symptoms on bronchial nonspecific responsiveness in rhinitic and asthmatic patients. The study was performed on 83 asthmatic patients and on 46 patients with allergic rhinopathy. The beginning of the symptoms and years of asthmatic or rhinitic history were particularly investigated. A histamine challenge was performed. The dose of histamine producing at 20% change in FEV1 (forced expiratory volume in one second) was calculated from the individual semilogarithmic dose-response curve (PD20). Bronchial responsiveness to histamine showed wide variability in subjects of two groups, and an overlap of the distribution curves was observed between asthmatic and rhinitic patients. A significant relationship (p less than 0.01) between the years elapsed from the onset of symptoms and bronchial responsiveness to histamine was observed in each group of patients. We noticed that the number of the years passed heightened the bronchial responsiveness to histamine in both groups of patients.

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.