Study of the role of the covalently linked cell wall protein (Ccw14p) and yeast glycoprotein (Ygp1p) within biofilm formation in a flor yeast strain.

Flor yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes-codifying for two cell surface glycoproteins-in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14).

Genomic signatures of adaptation to wine biological ageing conditions in biofilm-forming flor yeasts.

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

Thalidomide and sensory neurotoxicity: a neurophysiological study.

BACKGROUND: Recent studies confirmed a high incidence of sensory axonal neuropathy in patients treated with different doses of thalidomide. The study's aims were to measure variations in sural nerve sensory action potential (SAP) amplitude in patients with refractory cutaneous lupus erythematosus (CLE) treated with thalidomide and use these findings to identify the neurotoxic potential of thalidomide and the recovery capacity of sensory fibres after discontinuation of treatment. PATIENTS AND METHODS: Clinical and electrophysiological data in 12 female patients with CLE during treatment with thalidomide and up to 47 months after discontinuation of treatment were analysed. Sural nerve SAP amplitude reduction > or =40% was the criteria for discontinuing therapy. RESULTS: During treatment, 11 patients showed a reduction in sural nerve SAP amplitude compared to baseline values (9 with a reduction > or =50% and 2 <50%). One patient showed no changes in SAP amplitude. Five patients complained of paresthesias and leg cramps. After thalidomide treatment, sural SAP amplitude recovered in 3 patients. At detection of reduction in sural nerve SAP amplitude, the median thalidomide cumulative dose was 21.4 g. The threshold neurotoxic dosage is lower than previously reported. CONCLUSIONS: Sural nerve SAP amplitude reduction is a reliable and sensitive marker of degeneration and recovery of sensory fibres. This electrophysiological parameter provides information about subclinical neurotoxic potential of thalidomide but is not helpful in predicting the appearance of sensory symptoms.

Correlation between cell lipid content, gene expression and fermentative behaviour of two Saccharomyces cerevisiae wine strains.

AIM: To verify a possible correlation between cell lipid composition, expression of key genes in lipid metabolism and fermentative behaviour of Saccharomyces cerevisiae wine strains. METHODS AND RESULTS: The fermentative abilities of two commercial wine strains of S. cerevisiae were tested under stressful conditions. Cell number, glucose and fructose concentrations, expression of ACS1, ACS2, ACC1, OLE1, ERG9, ERG10, ARE1 and ARE2 and lipid content were evaluated. The strain that failed to complete the fermentation had lower amounts of C16:1 and C16:0 fatty acids at the beginning of fermentation (0 h) and late logarithmic phase (72 h). While the amount of C18:1 in this strain was lower than that in the strain that completed the fermentation at 0 h, same levels were observed for both strains at 72 h. The sterol levels were generally higher in the strain that failed to complete the fermentation. Gene expression generally increased from the beginning of the fermentation to the late logarithmic phase in both strains. CONCLUSION: A positive correlation between good fermentative ability, elevated fatty acid content and ACC1 gene expression has been identified. SIGNIFICANCE AND IMPACT OF THE STUDY: The cell lipid content at the time of inoculum and expression of ACC1 gene of starter strains should be carefully considered in order to identify the possible stuck/sluggish fermentations.

Positive and negative effects of thalidomide on refractory cutaneous lupus erythematosus.

BACKGROUND: Thalidomide is used in cutaneous lupus erythematosus (CLE) refractory to conventional therapies. Peripheral neuropathy (PN) is the most severe side effect, but the incidence of PN and its relation to thalidomide dose are still unclear. OBJECTIVE: To prospectively evaluate the efficacy as well as the occurrence of PN in CLE patients treated with thalidomide, and to assess whether PN, when occurs, correlates with thalidomide dose and/or length of treatment. METHODS: Fourteen female patients with CLE in low-dose thalidomide therapy were followed for up to 24 months. Prior to, and regularly during treatment patients underwent rheumatological, dermatological, neurological and electrophysiological evaluations. A decline in sural SNAP of 50% or more from baseline value was considered as criterion of sensory axonal PN. RESULTS: All patients showed a dramatic improvement of skin manifestations. Ten patients (71.4%) developed a sensory axonal PN. The median time free from this complication was 14 months. No correlations were found between age of the patients nor thalidomide cumulative dose and occurrence of PN (Mann-Whitney U Test; p>0.16). Other adverse effects were: tremor, paresthesias, somnolence, amenhorrea, constipation and thoracic pain. CONCLUSIONS: Low does thalidomide is efficacious in treating CLE, but PN is a common complication whose occurrence does not seem to correlate with total thalidomide dose, whereas with the duration of therapy. A closer electrophysiological follow-up is therefore recommended in the long-term treatment.

Neurological complications of celiac disease and autoimmune mechanisms: preliminary data of a prospective study in adult patients.

Antibodies to gangliosides and Purkinje cells have been reported in patients with celiac disease (CD) with neuropathy and ataxia, respectively. Whether these antibodies are pathogenic is not clear. The response of neurological symptoms and antibody titers to a gluten-free diet is still controversial. The objective of our study was to assess whether neurological manifestations in CD patients correlate with antibody titers and a gluten-free diet.Thirty-five CD patients (9 males, 26 females, mean age 37.1 +/- 12.6 yrs) were followed prospectively. At initial evaluation, 23 were on a gluten-free diet, 12 were not. At recruitment and during follow-up, patients underwent neurological and electrophysiological evaluation. IgG, IgM, and IgA anti-ganglioside antibodies were assayed by ELISA; anti-neuronal antibodies were assessed by immunohistochemistry and Western blot. Four patients, all males, had electrophysiological evidence of neuropathy; three had been on a gluten-free diet for several months, and one was newly diagnosed. One had reduced tendon reflexes; another complained of distal paresthesias. With regard to anti-ganglioside antibodies, three patients had a moderate increase in antibodies without symptoms or signs of neuropathy. No patients had ataxia or cerebellar dysfunction, although in four patients reactivity to neuronal antigens was found. In 17 patients, an electrophysiological follow-up (mean duration of follow-up, 9 months) showed no changes. In conclusion, the preliminary results of this prospective study indicate that neuropathy, usually subclinical, may accompany CD. Antibody titers do not seem to correlate with neurological symptoms/signs or diet. Ongoing follow-up will help confirm these data and clarify the role, if any, of antibodies in neurological involvement in CD.

Long-term analgesic effect of clodronate in rodents.

Several studies have shown that treatment with bisphosphonates can reduce the pain associated with different painful diseases. In a previous study we demonstrated that in mice two bisphosponates, clodronate and pamidronate, had an antinociceptive effect under acute conditions not related to bone processes, after in vein (iv) or intracerebroventricular (icv) injection. The present study tested the time-dependent antinociceptive action of clodronate and pamidronate in comparison with that of acetylsalicylic acid (ASA) and morphine after iv and icv injection using the tail-flick test in acute and chronic treatment. The effects of clodronate on other measures of animal behaviour were also evaluated. In the tail-flick test, administration of clodronate iv produced an antinociceptive effect that was greater than that of ASA and statistically significant up to 16 h; pamidronate iv showed a significant antinociceptive effect for only 6 h. Clodronate and pamidronate icv showed an increase in tail-flick latency time that was significant and lasted for 16 and 6 h, respectively, while morphine produced an antinociceptive effect for 24 h. In the test we found significant differences between male and female mice in the latency time values but not in the length of the analgesic effect. In the chronic treatment paradigm, clodronate produced a significant increase of the tail-flick latency after the first injection. The analgesic effect increased up to 50% after 5 days of treatment. Significant analgesic effects were still present after 3, 7, and 14 days from the end of treatment. Clodronate did not produce any significant behavioural effects in the Rota-rod test, pentobarbital-induced sleeping time, and locomotor activity cage. These data indicate that clodronate presents a central and peripheral prolonged antinociceptive effect, without any behavioural side effects.

Analgesic effect of bisphosphonates in mice.

Bisphosphonates are analogues of inorganic pyrophosphate and are inhibitors of bone resorption. Many derivatives have been developed for the treatment of enhanced bone resorption; several reports reveal that treatment with bisphosphonates is able to reduce the pain associated with different painful diseases. This study tested the antinociceptive action of four bisphosphonates, clodronate, alendronate, pamidronate and etidronate, in comparison with that of morphine and acetylsalicylic acid using two algesimetric tests in mice, tail-flick and writhing tests. In the tail-flick test, after intravenous (i.v.) injection, a dose-dependent antinociception was present after pamidronate, clodronate and acetylsalicylic acid whereas etidronate and alendronate produced an analgesic effect only with the highest dose tested. We also studied the central effect of clodronate and pamidronate and, after intracerebroventricular injection, both bisphosphonates showed a dose-dependent antinociceptive effect. In the writhing test clodronate and pamidronate showed a statistically significant antinociceptive action after i.v. and intramuscular administration. To verify if clodronate and pamidronate could modulate the peripheral opioid receptors we evaluated the gastrointestinal transit time in mice, but we did not find any effect on the gastrointestinal motility. These data indicate that clodronate and pamidronate present a central and peripheral antinociceptive effect; however, the main mechanism cannot be determined from the present data. We discuss the possible pharmacological hypothesis to interpret the present results. The findings suggest a pharmacological role of the bisphosphonates in the modulation of antinociception even in acute conditions not related to accelerated osteolytic and inflammatory response, with a possible clinical application to control pain.

Chlorpromazine disposition in relation to age in children.

The pharmacokinetics of chlorpromazine after intravenous infusion were studied in 25 children. The pharmacokinetic parameters studied are markedly different from those reported for adults. A clear relationship was demonstrated between age, serum terminal half-life (r = 0.75) and systemic clearance (r = -0.43). It appears that the pharmacokinetics of chlorpromazine are more rapid in children than in adults.

Anti-ganglioside antibodies in children with coeliac disease: correlation with gluten-free diet and neurological complications.

BACKGROUND: Emerging evidence points to humoural mechanisms in neurological complications of coeliac disease. Immunoglobulin G anti-ganglioside antibodies have been reported in coeliac disease patients with neuropathy, suggesting an immune response to peripheral nerve antigens. No data are so far available on anti-ganglioside antibodies in coeliac disease children or on antibody modifications after gluten-free diet. AIM: To evaluate the presence of antibodies to ganglioside antigens in children with coeliac disease, their modification after gluten-free diet, and possible correlations with neurological manifestations. METHODS: Sera from 42 coeliac disease children, before and after gluten-free diet, were tested by enzyme-linked immunosorbent assay for the presence of antibodies (immunoglobulin M, immunoglobulin A, immunoglobulin G) to gangliosides. Thirty-five sera of age-matched children with dyspepsia were used as control. RESULTS: High anti-ganglioside antibodies titres were present in two patients. In one patient, antibody titre reversed after gluten-free diet, whereas in the other one the titre increased after diet. Neither one complained of neurological symptoms. CONCLUSIONS: Anti-ganglioside antibodies do not seem to correlate with gluten ingestion or with neurological manifestations in children with coeliac disease. Mechanisms different from gluten exposure may be implicated in the antibody production. An ongoing prospective study will help clarify the role, if any, of these antibodies in coeliac disease.

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study.

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine. The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively. The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.

Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line.

Solid lipid nanoparticles (SLN) carrying cholesteryl butyrate (chol-but), doxorubicin and paclitaxel had previously been developed, and the antiproliferative effect of SLN formulations versus conventional drug formulations was here evaluated on HT-29 cells. The 50% inhibitory concentration (IC(50) values were interpolated from growth curves obtained by trypan blue exclusion assay. In vitro cytotoxicity of SLN carrying chol-but (IC(50 72 h) 0.3 +/- 0.03 mM vs >0.6 mM) and doxorubicin (IC(50 72 h) 81.87 +/- 4.11 vs 126.57 +/- 0.72 nM) was higher than that of conventional drug formulations. Intracellular doxorubicin was double after 24 h exposure to loaded SLN versus the conventional drug formulation, at the highest concentration evaluated by flow cytometry. In vitro cytotoxicities of paclitaxel-loaded SLN and conventional drug formulation (IC(50 72 h) 37.36 +/- 6.41 vs 33.43 +/-1.17 nM) were similar. Moreover, the combination of low concentrations of chol-but SLN (0.1-0.2 mM) and doxorubicin (1.72 nM) or paclitaxel (1.17 nM) exerted a greater-than-additive antiproliferative effect at 24 h exposure, while the combination of Na-but and doxorubicin or paclitaxel did not. These preliminary in vitro results suggest that SLN could be proposed as alternative drug delivery system.

Biodistribution of stealth and non-stealth solid lipid nanospheres after intravenous administration to rats.

Drug-free stealth and non-stealth solid lipid nanospheres (SLNs) were administered intravenously to rats to evaluate their tissue distribution and their transport across the blood-brain barrier. Two types of experiments were performed using unlabelled and labelled SLNs. Rats were administered labelled non-stealth or stealth nanospheres (NSSLNs and SSLNs) and their tissue distribution was monitored for 60 min. In another experiment, rats were injected with unlabelled NSSLNs or SSLNs and the cerebrospinal fluid (CSF) was analysed using transmission electron microscopy (TEM) to confirm the presence of the SLNs. Some differences were found in the biodistribution between labelled NSSLNs and SSLNs. In particular, the radioactivity in the liver and the lung was much lower for SSLNs than for NSSLNs, confirming a difference in their uptake. Both types of SLNs were detected in the brain. TEM analysis showed both types of SLNs in rat CSF.

Imipramine pharmacokinetics in depressed geriatric patients.

Pharmacokinetics of single dose (25 mg i.m.) of imipramine was studied in geriatric and young adult patients with mood disorders requiring antidepressant treatment. Imipramine and its principal metabolites were determined by means of a high performance liquid chromatography method with electrochemical detection. Serum concentrations analysis was performed according to a bicompartmental open model. A relationship between age and kinetic parameters such as area under the curve, elimination phase constant, half-life and total body clearance was observed. Significant differences were demonstrated between elderly and young adult patients when comparing these parameters. It is concluded that geriatric patients treated with imipramine should receive doses of about 60% of those of young adults.

Conduction studies as prognostic parameters in the natural history of diabetic neuropathy: a long-term follow-up of 114 patients.

In order to evaluate the clinical and prognostic role of common neurophysiological parameters, we examined 114 patients selected from a population of 2500 diabetics, observed in the period 1973-1986. The follow-up lasted 9 to 16 years (mean = 10). For prognostic purposes the patients were divided into 3 groups according to the motor conduction velocity (MCV) of the peroneal nerve: A) 47 cases with normal MCV (more than 44 m/s); B) 38 cases with mild neuropathy (MCV between 44 and 39 m/s); C) 29 cases with severe neuropathy (MCV less than 39 m/s). The natural history was evaluated on the basis of the following parameters: neurological symptom score (NSS), neurological disability score (NDS), patient's self-evaluation (PE) and neurologist's evaluation (ME). At the initial examination, there was a significant correlation between MCV and NDS/ME. At the final examination, MCV was reduced to groups A and B, unchanged in group C. NSS, NDS, PE and ME were worsened, but a significant correlation was found only between MCV and NDS: neuropathic groups (B and C) showed a more severe evolution than the normal group (A). In conclusion, a) MCV is progressively reduced as diabetes evolves, but--once it has reached a plateau--it stabilizes; b) MCV only has a prognostic value when it is considered together with clinical neurological signs.

[The lower esophageal sphincter in gastrectomized patients. Manometric study]. / Lo sfintere esofageo inferiore nel gastrooresecato. Studio manometrico.

New manometric techniques in for examining the lower oesophageal sphincter (LOS) were applied an investigation of the oesophago-gastric junction after partial gastric resection. Pressure and blood gastrin data are reported for eight cases examined before and after surgery, under basal conditions and after stimulation with a protein meal. It was found that gastric resection leads to a decrease in LOS performance (43.6% fall in maximum pressure) and length (-33.3%). There is also a 93.5% decrease in the pressure response to a protein meal, and hence a predisposition to gastroesophageal reflux.

[Long-term effects of gastric resection on the inferior esophageal sphincter]. / Effetti a distanza della resezione gastrica sullo sfintere esofageo inferiore.

The manometric profile of the oesophagogastric junction has been studied in patients subjected one year earlier to partial gastric resection. A reduction in maximum basal pressure (--49.8%) and length of the lower oesophageal sphincter (--35.45%) were noted. These figures agree with what was observed one month after operation in a previous study. It is concluded that gastric resection lead to a non-transitory reduction in sphincter function.

Economic impact of resistance in the community.

Antimicrobial resistance is assumed to be an important health problem and an economic burden to society. However, the relationship between the emergence of in vitro microbiological resistance and its clinical and socioeconomic consequences has not yet been satisfactorily determined for either nosocomial or community-acquired infection. In the case of both nosocomial and community-acquired infection, previous exhaustive reviews of published and unpublished reports concluded that mortality, likelihood of hospitalization, and length of hospital stay were usually at least twice as great for patients infected with drug-resistant strains as for those infected with drug-susceptible strains of the same bacteria. However, evaluation of the economic impact of resistance is still problematic and the adverse economic and health effects of drug-resistant bacterial infections can only be roughly quantified. Decision analysis models, such as decision trees, can aid evaluation of the impact of resistance on health and economic outcomes from the perspective of a given decision maker. A model of cost analysis should be based on a knowledge of the incremental consumption of resources specifically dependent on the dynamics of antimicrobial resistance in a given clinical setting (e.g. home care or hospital care). In general, we can assume that the increased rate of isolation of resistant strains from community-acquired infections correlates positively with an increase in morbidity, mortality, risk of hospitalization and the need for additional days in hospital and for more expensive and powerful antibiotics. We implemented and simulated a general decision-tree model to analyse the influence of antibiotic resistance on the economic outcomes of community-acquired lower respiratory tract infections, from the perspective of both society and the health-care local organization (HCLO). This model allows simulation of the impact of different degrees of resistance on the direct costs of an antibiotic therapy as well as on the cost-effectiveness of antibiotics with different degrees of resistance.

Innovative formulations for the controlled and site-specific delivery of antiinflammatory drugs.

Pharmaceutical technology has introduced a promising pathway in the future of medicine in particular nanotechnological innovations have provided the opportunity to design and develop efficient drug delivery systems able to target and treat several diseases, including those mediated by inflammation. The engineering of drug delivery systems can be used to target tissues involved in the pathology under treatment, to avoid early drug biological environmental degradation and to modulate drug pharmacokinetics. Glucocorticoids and non-steroidal anti-inflammatory drugs are the most commonly prescribed drug categories worldwide for the treatment of disorders associated with inflammation. Although glucocorticoids can be highly effective in treating inflammation, their systemic application is limited due to the high incidence of serious adverse effects, mainly in long-term treatment. Non-steroidal anti-inflammatory drugs are a heterogeneous group of compounds and most of them have unfavorable pharmacokinetics and pharmacodynamics, leading to adverse effects, such as gastrointestinal disorders. Therefore, the need for drug delivery systems for long term administration of anti-inflammatory drugs with a well-controlled release profile is evident. The aim of this review is to assess innovative colloidal drugs carriers, in particular liposomes and nanoparticles, with special focus on site-specific delivery for particularly problematic tissues such as the gastrointestinal tract, joints and eyes.

Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models.

BACKGROUND AND PURPOSE: Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models. EXPERIMENTAL APPROACH: Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice. KEY RESULTS: Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth. CONCLUSION AND IMPLICATIONS: Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug.