A genome-wide nanotoxicology screen of Saccharomyces cerevisiae mutants reveals the basis for cadmium sulphide quantum dot tolerance and sensitivity.

The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.

Degradation of trans-ferulic and p-coumaric acid by Acinetobacter calcoaceticus DSM 586.

Cell suspensions of Acinetobacter calcoaceticus strain DSM 586 and DSM 590 were able to grow on benzoic, p-hydroxybenzoic and vanillic acid as sole carbon source. Testing the utilization of trans-ferulic and p-coumaric acid, we found that the sole A. calcoaceticus DSM 586 efficiently degraded the lignocellulose related monomers. Cells induced with trans-ferulic acid were able to oxidize trans-ferulic, p-coumaric, vanillic, p-hydroxybenzoic and protocatechuic acid at rates higher than the uninduced culture. The same activity was found in the p-coumaric acid induced culture. Two aromatic compounds, vanillic and p-hydroxybenzoic acid, were isolated from culture filtrates of trans-ferulic and p-coumaric acid grown cells, respectively, and further characterized by high performance liquid chromatography. 1H- and 13C-nuclear magnetic resonance and ultraviolet spectrophotometry. Cell extracts of trans-ferulic or p-coumaric acid induced cultures were shown to rapidly convert protocatechuic acid to beta-carboxymuconic acid. Moreover, A. calcoaceticus DSM 586 produced high levels of protocatechuic 3,4-dioxygenase compared to cathecol 1,2-dioxygenase and gentisate 1,2-dioxygenase in the degradation of trans-ferulic or p-coumaric acid. Based upon these results, a reaction sequence for the complete degradation of trans-ferulic and p-coumaric acid in A. calcoaceticus DSM 586 is proposed.

Characterization of microbial cellulose from a high-producing mutagenized Acetobacter pasteurianus strain.

A wild-type Acetobacter pasteurianus was subjected to chemical mutagenesis for the induction and isolation of a cellulose overproducing strain. A mutagenized strain capable of synthesizing double amounts of cellulose compared to the wild type was obtained. Cellulose, both from the wild-type and the mutagenized strain, was extracted and purified for chemical characterization and investigation of its physico-chemical properties. The comparison of the two microbial polysaccharides shows that the putative mutation of A. pasteurianus strain had no effect on some cellulose features such as chemical structure, polymorphic form, crystallinity.

Nutritional status and airflow obstruction: two independent contributors to CO diffusing capacity impairment in COPD.

BACKGROUND: The association between weight loss and Chronic Obstructive Pulmonary Disease (COPD) has been recognised from many years. Based on the evidence that nutritional status reflects metabolic disturbances in COPD, the relationship between body mass index (BMI), severity of airflow obstruction and CO diffusing capacity (DL(CO)), that is the functional hallmark of emphysema, is relevant to the management of COPD phenotypes. METHODS: We reviewed 104 patients with COPD (82 males), aged 66 +/- 9 years (mean +/- SD). Height averaged 165 +/- 8 cm, weight 71 +/- 16 Kg, FEV1 50 +/- 18 (% of predicted), RV 169 +/- 49%, and DL(CO) 56 +/- 26%. Multiple linear regression was performed using BMI, FEV1 and RV, as explanatory variables for DL(CO). Patients were also classified into four groups according to BMI < or = 18.5 (low), > 18.5 and < or = 25 (ideal), > 25 and < or = 30 (overweight), > 30 (obese), and post-bronchodilator FEV1 < 50%. Using this categorisation, a two-factor analysis of variance, testing for interaction and main effects (BMI and FEV1) was performed as confirmatory analysis for the association between BMI (kg/m2), FEV1% and DL(CO)%. RESULTS: FEV1 and BMI were significantly and independently associated to DL(CO) according to the equation: DL(CO) = -18.32 + 0.65 x FEV1 + 1.59 x BMI (R2 = 0.40, p < 0.0001). The contribution of RV % to DL(CO) % was largely non-significant (p = 0.16). A close relationship was found between BMI (kg/m2) and DL(CO) %, for all of the four BMI groups segregated by post-bronchodilator FEV1 %, (p < .0001). No interaction was found between these two factors (p = 0.30). CONCLUSION: Nutritional status as assessed by BMI contributes substantially to impairment of DL(CO) independently of the severity of airflow obstruction. This data confirms the association between emphysematous process and weight loss in advanced COPD, independent of the airflow obstruction severity.

Molecular engineering with the FRT sequence of the yeast 2 microm plasmid: [cir0] segregant enrichment by counterselection for 2 microm site-specific recombination.

Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.

High-level heterologous gene expression in Saccharomyces cerevisiae from a stable 2 microns plasmid system.

The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2 microns plasmid. Nevertheless, derivatives of the 2 microns plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells. We report the construction and utilization of an efficient heterologous gene expression system containing a 4.5-kb inducible expression cassette inserted into the 2 microns plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination. The non-selectable 2 micron plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number. The dynamics of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions. The heterologous lacZ reporter gene coding for beta-galactosidase (beta Gal) is driven by the hybrid, galactose-inducible promoter GAL10::pMF alpha 1. Upon induction, beta Gal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium. The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MF alpha 1 signal sequence, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae.

The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.

Effects of celiprolol on the bronchial reactivity in asthma.

In 10 patients with bronchial asthma but normal ventilatory function, celiprolol, a cardioselective beta-adrenoreceptor antagonist, did not significantly affect forced expiratory volume in 1 second (FEV1) or airways resistance (Raw). In contrast, metoprolol substantially reduced FEV1 and increased Raw. In addition, compared with metoprolol, celiprolol induced a greater recovery of FEV1 and Raw after methacholine-induced bronchoconstriction.

Erectile dysfunction in men with obstructive sleep apnea: an early sign of nerve involvement.

Erectile dysfunction (ED) is common in men with obstructive sleep apnea (OSAS) but no completely convincing hypotheses about the underlying pathogenic mechanisms have been published in the literature. The aims of the present study were to assess the presence of ED in a group of OSAS patients without daytime respiratory failure and to determine whether this dysfunction was related to peripheral nerve involvement. Evaluation of the bulbocavernosus reflex (BCR) and the somato-sensory evoked potentials of pudendal nerve (PSEPs), the most widely established method of documenting pudendal neuropathies as being the cause of impotence, was performed in 25 patients. Data on BCR were compared with those of 25 healthy males volunteers matched for age. BCR was altered in 17 patients: in 6 it was elicited while in 11 it had a prolonged latency and reduced amplitude. Patients with altered BCR presented an higher AHI, an higher percentage of sleep time spent with SaO2 <90% (TST90) and a lower daytime PaO2. Six patient had clinically silent neurophysiological signs of mild polyneuropathy. The degree of OSAS and gas exchange alteration was more severe in patients with polyneuropathy than in those with isolated BCR alteration. ED is a common finding in OSAS patients and this alteration seems to be related to a nerve dysfunction. The development of nerve dysfunction is associated with a more severe degree of OSAS and nocturnal hypoxia.

Distribution of bronchial nonspecific reactivity in the general population.

We investigated 654 subjects of a small Lombardy (Italy) town between 15 and 64 years of age who were representative of the general population. By clinical examination, the sample included 535 normal subjects (164 normal smokers, 341 normal nonsmokers, 30 normal subjects with acute upper respiratory illness within 30 days before the challenge), 50 with chronic bronchitis, 26 with asthma, and 43 with allergic rhinitis. Subjects whose FEV1 was 75 percent or more than the predicted value (654) underwent methacholine bronchial challenge by means of 1 percent metered-dose solution. The test result was considered positive at a drop of more than 15 percent in FEV1 (compared with buffer). Normal smokers and all of the groups with disease had a significantly different distribution of reactivity compared with normal nonsmokers. The difference between asthmatic and these "normal" subjects was highly significant; nevertheless, a clear cut-off between the two groups does not appear to exist.

The development of hyperventilation in patients with chronic heart failure and Cheyne-Strokes respiration: a possible role of chronic hypoxia.

AIM: To analyze the relationship between daytime respiratory and cardiac function in patients with compensated chronic heart failure (CHF) with and without periodic breathing (PB) or Cheyne-Stokes respiration (CSR). PATIENTS: We studied 132 patients (female, 13%; mean age, 53+/-8 years; body mass index, 25.9+/-3.5 kg/m2; left ventricular ejection fraction <40%; 23% in New York Heart Association class I, 43% in class II, and 34% in class III-IV). METHODS: Measurement of pulmonary function and blood gases, hemodynamic evaluation, analysis of breathing profile, echocardiography, recording of ECG, beat-to-beat arterial oxygen saturation, and respiration during spontaneous breathing. RESULTS: Fifty-eight percent of patients showed PB or CSR. Patients with PB or CSR have greater cardiac function impairment. Mean values of lung volumes and PaO2 were similar in the three groups of patients considered. In contrast, patients with PB or CSR had an increased minute ventilation and reduced PaCO2 values. Interestingly, patients with PB or CSR had lower values of arterial content of O2 and systemic oxygen transport (SOT) than patients with a normal breathing pattern (SOT, 394+/-9.8, 347+/-9.6, 438+/-11 mL of O2/min/m2, respectively; analysis of variance p<0.001). Weak correlations were found among lung volumes, blood gases, and cardiac function parameters: ie, vital capacity was correlated inversely with pulmonary capillary wedge pressure (PCWP) (-0.25; p<0.05); PaCO2 with PCWP (r=0.26; p<0.05), lung-to-ear circulation time (LECT) (r=-0.4; p<0.05), SOT (r=-0.33; p<0.0001), and cardiac index (CI) (r=0.27; p=0.003). Multiple regression analyses showed that arterial PCO2 was significantly correlated with SOT, LECT, and CI (r=0.51; r2=0.26; p<0.000001); the correlation became stronger considering only CSR patients (r=0.64; r2=0.4; p<0.001). CONCLUSIONS: Our study shows that patients with daytime breathing disorders have chronic hypocapnia. A reduced SOT may be one of the stimuli determining increased minute ventilation in these patients.

Respiratory response and inspiratory effort during pressure support ventilation in COPD patients.

OBJECTIVE: Pressure Support Ventilation (PSV) is now widely used in the process of weaning patients from mechanical ventilation. The aim of this study was to evaluate the effects of various levels of PS on respiratory pattern and diaphragmatic efforts in patients affected by chronic obstructive pulmonary disease (COPD). SETTING: Intermediate intensive care unit. PATIENTS: We studied ten patients undergoing PSV and recovering from an episode of acute respiratory failure due to exacerbation of COPD. METHODS: Three levels of PSV were studied, starting from the lowest (PSb) one at which it was possible to obtain an adequate Vt with a pH > or = 7.32 and an SaO2 > 93%. Then, PS was set at 5 cmH2O above (PSb + 5) and below (PSb-5) this starting level. Ventilatory pattern, transdiaphragmatic pressure (Pdi), the pressure-time product of the diaphragm (PTPdi), the integrated EMG of the diaphragm, static PEEP (PEEPi, stat), dynamic PEEP (PEEPi, dyn), and the static compliance and resistance of the total respiratory system were recorded. RESULTS: Minute ventilation did not significantly change with variations in the level of PS, while Vt significantly increased with PS (PS-5 = 6.3 +/- 0.5 ml/kg vs. PSb = 10.1 +/- 0.9 [p < 0.01] and vs. PS + 5 = 11.7 +/- 0.6 [p < 0.01]), producing a reduction in respiratory frequency with longer expiratory time. The best values of blood gases were obtained at PSb, while at PSb-5, PaCO2 markedly increased. During PSb and PSb + 5 and to a lesser extent during PSb-5, most of the patients made several inspiratory efforts that were not efficient enough to trigger the ventilator to inspire; thus, the PTPdi "wasted" during these inefficient efforts was increased, especially during PS + 5. The application of an external PEEP (PEEPe) of 75% of the static intrinsic PEEP during PSb caused a significant reduction in the occurrence of these inefficient efforts (p < 0.05). Minute ventilation remained constant, but Vt decreased, together with Te, leaving the blood gases unaltered. The PTPdi per breath and the dynamic PEEPi were also significantly reduced (by 59% and 31% of control, respectively, p < 0.001) with the application of PEEPe. CONCLUSION: We conclude that in COPD patients, different levels of PSV may induce different respiratory patterns and gas exchange. PS levels capable of obtaining a satisfactory equilibrium in blood gases may result in ineffective respiratory efforts if external PEEP is not applied. The addition of PEEPe, not exceeding dynamic intrinsic PEEP, may also reduce the metabolic work of the diaphragm without altering gas exchange.

Elevated static compliance of the total respiratory system: early predictor of weaning unsuccess in severed COPD patients mechanically ventilated.

OBJECTIVE: To assess in a group of COPD patients mechanically ventilated for an episode of acute respiratory failure the respiratory mechanics with a simple and non invasive method at the bedside in order to evaluate if these parameters may be predictive of weaning failure or success. DESIGN: A prospective study. SETTING: Intensive care and intermediate intensive care units. PATIENTS: 23 COPD patients ventilated for acute respiratory failure and studied within 24 hours from intubation. METHODS: Using end-expiratory and end-inspiratory airway occlusion technique, we measured PEEPi, static compliance of the respiratory system (Crs, st) maximum respiratory resistance (Rrsmax) and minimum respiratory resistance (Rrsmin). MEASUREMENTS AND RESULTS: The weaned group (A) and the not weaned group (B) were not different regarding to static PEEPi (group A 8.5 +/- 4.0 vs group B 8.9 +/- 2.6 cmH2O), TO Rrsmax (22.4 +/- 5.3 versus 22.2 +/- 9.0 cmH2O/1/s) and to Rrsmin (17.6 +/- 5.5 versus 17.9 +/- 8.0 cmH2O/1/s), while a significant difference (p < 0.001) has been found in Cst, rs (62.7 +/- 17.% versus 111.6 +/- 18.0 ml/cm H2O). The threshold value of 88.5 ml/cmH2O was identified by discriminant analysis and provided the best separation between the two groups, with a sensitivity of 0.85 and a specificity of 0.87. CONCLUSION: Cst, rs measured non invasively in the first 24 h from intubation, provided a good separation between the patients who were successfully weaned and those who failed.

Breathing patterns and HbSaO2 changes during nocturnal sleep in patients with Duchenne muscular dystrophy.

A night-time polygraphic sleep recording with continuous HbSaO2 monitoring was performed in 11 chair-bound Duchenne muscular dystrophy patients with severe restrictive lung disease but with blood gas values within normal limits when awake. No abnormalities of sleep pattern were detected. Nocturnal sleep did not have significant adverse effects on respiration. However, in 6 patients, infrequent central apnoeas or hypopnoeas occurred which were associated with falls in HbSaO2 greater than those that have been reported to be in normal subjects. The magnitude of HbSaO2 falls appeared to be significantly correlated with functional residual capacity values. Overall, the findings revealed a relatively preserved, although unstable, blood O2 balance during nocturnal NREM and REM sleep in patients with Duchenne muscular dystrophy, even in an advanced stage of their illness.

A clinical, EEG and CT study in 21 cases of chronic respiratory failure.

A clinical, EEG and CT study was carried out on 21 patients with chronic stable respiratory failure. The neurological disturbances and mental deterioration observed were of a mild degree, and no severe alterations were detected at EEG. CT showed cerebral atrophy, but the type and prevalence did not differ substantially between patients studied and normal sex- and age-matched control subjects. The data gave evidence that in chronic stable respiratory failure cerebral function is only slightly affected.

HIV infection and suicidality.

Based on the published literature of the past 10 years, this is a review concerning the topic of HIV-spectrum pathology and suicidality from a markedly suicidological point of view. HIV infection is considered as a possible suicide risk factor among several other ones involved. We distinguish between research on complete suicide, attempted suicide, DSH, and suicidal thoughts. We have analytically examined the overlapping of the complex area of HIV-infectious pathology and of the heterogenous area of suicide behaviour: a unifying picture may be achieved by introducing the concept of 'mosaicism'.

Analysis of variance in determinations of equivalence volume and of the ionic product of water in potentiometric titrations.

Homogeneous sets of data from strong acid-strong base potentiometric titrations in aqueous solution at various constant ionic strengths have been analysed by statistical criteria. The aim is to see whether the error distribution matches that for the equilibrium constants determined by competitive potentiometric methods using the glass electrode. The titration curve can be defined when the estimated equivalence volume VEM, with standard deviation (s.d.) sigma (VEM), the standard potential E(0), with s.d. sigma(E(0)), and the operational ionic product of water K(*)(w) (or E(*)(w) in mV), with s.d. sigma(K(*)(w)) [or sigma(E(*)(w))] are known. A special computer program, BEATRIX, has been written which optimizes the values of VEM, E(0) and K(*)(w) by linearization of the titration curve as a Gran plot. Analysis of variance applied to a set of 11 titrations in 1.0M sodium chloride medium at 298 K has demonstrated that the values of VEM belong to a normal population of points corresponding to individual potential/volume data-pairs (E(i); v(i)) of any titration, whereas the values of pK(*)(w) (or of E(*)(w)) belong to a normal population with members corresponding to individual titrations, which is also the case for the equilibrium constants. The intertitration variation is attributable to the electrochemical component of the system and appears as signal noise distributed over the titrations. The correction for junction-potentials, introduced in a further stage of the program by optimization in a Nernst equation, increases the noise, i.e., sigma(pK(*)(w)). This correction should therefore be avoided whenever it causes an increase of sigma(pK(*)(w)). The influence of the ionic medium has been examined by processing data from acid-base titrations in 0.1M potassium chloride and 0.5M potassium nitrate media. The titrations in potassium chloride medium showed the same behaviour as those in sodium chloride medium, but with an s.d. for pK(*)(w) that was smaller and close to the expected instrumental noise, whereas the titrations in nitrate medium had a high noise level and even the determination of VEM was less certain. Procedures are also proposed for obtaining reference sets of data and checking the conformity of the solutions and apparatus to the chosen reference.

Fractal properties of DNA walks.

We describe two dimensional DNA walks, and analyze their fractal properties. We show results for the complete genome of S. cerevisiae. We find that the mean square deviation of the walks is superdifussive, corresponding to a fractal structure of dimension lower than two. Furthermore, the coding part of the genome seems to have smaller fractal dimension, and longer correlations, than noncoding parts.

Chronic respiratory failure in a patient with type I Arnold-Chiari malformation (ACM1) and syringomyelia.

We present the case of a young adult with type I Arnold-Chiari malformation (AMC1) and syringomyelia who developed central sleep apnoea and chronic respiratory failure, successfully treated with nocturnal noninvasive positive pressure ventilation ventilation (NIPPV). An extensive syringomyelic cavity (from bulbar to L4 segment) with severe impairment of the IX cranial nerve was documented and remains, although reduced, after the neurosurgical treatment. At baseline evaluation, the patient showed a moderate restrictive ventilatory defect, severe hypercapnic respiratory failure, abnormal control of breathing characterized by the absence of response to hypoxia and hypercapnia, and severe nocturnal central apnoeas. Nocturnal NIPPV was then started in the A/C mode with an improvement in blood gas values. Further evaluations were performed 10 and 18 months later. A progressive significant improvement of lung volumes, both in sitting and supine position, associated with a slight improvement of blood-gas values were observed. Nonetheless, the breathing pattern abnormalities persisted. Polysomnographic evaluation during mechanical ventilation showed a normalization of breathing pattern with arterial oxygen saturation (SaO2) > 90% throughout the night.

Chronic respiratory symptoms, bronchial responsiveness and dietary sodium and potassium: a population-based study.

A possible relationship between Na+ intake and increased prevalence and mortality from asthma and chronic obstructive pulmonary disease (COPD) has been suggested but not clearly proven for several reasons (difficulty in assessing Na+ and K+ both by 24 h excretion and dietary recall, too small an effect of these ions on the pathology, and the role of potential confounders). We wanted to determine the relationship of Na+ and K+ intake, assessed by means of a 7 day recall, with chronic respiratory symptoms and bronchial responsiveness in a sample of the general population. Two hundred and five subjects were studied, with complete dietary and respiratory questionnaires, and baseline respiratory function tests, together with a subsample of 146 subjects who underwent histamine challenge. The 7 day recall consisted of two parts: the first assessed discretionary Na+; and the second assessed Na+ and K+ contained in food. The whole sample was split into two groups based on the levels of consumption, and the statistical analysis was performed contrasting the three lower quartiles vs the highest. Smoking habit, social economic status, age and body mass index (BMI) were not confounders for Na+ and K+ intake. The prevalence of symptomatic subjects and baseline respiratory function values were not significantly different in the two groups of quartiles for Na+ and K+. Baseline respiratory values and dose-response slope of the subsample were also not significantly different. We did not prove a relationship between these dietary factors and either bronchial responsiveness or chronic respiratory symptoms. Although we consider that our questionnaire is more reliable than other methods for Na+ and K+ assessment, several potential biases still remain.(ABSTRACT TRUNCATED AT 250 WORDS)