Characterisation of EFV12 a bio-active small peptide produced by the human intestinal isolate Lactobacillus gasseri SF1109.

EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties are characterised. In particular, we propose a possible mechanism of action for EFV12 involving bacterial membranes targeting. Moreover, we show that this small peptide is able to bind lipopolysaccharides (LPS) and to counteract its inflammatory insult preventing LPS action on Toll-like receptor 4, thus interfering with extracellular signal-regulated kinase, p38 and Jun N-terminal kinase, mitogen-activated protein kinases signalling pathways. Altogether these observations suggest that the bioactive peptide EFV12 is a good candidate to promote L. gasseri induced gut homeostasis and counteracting intestinal pathogens.

Allergic and nonallergic rhinitis and skin sensitization to metals: is there a link?

SUMMARY: Background. Chromium, Cobalt and Nickel are responsible for contact dermatitis, that is largely prevalent in the general population. They can act also as irritants in the upper and lower respiratory airways. Also rhinitis (allergic and nonallergic) is a high prevalence disorder. Both diseases could share some common inflammatory mechanisms, but the clinical association between skin sensitization to metals and rhinitis was never studied. Objective. We assessed the presence of skin sensitization to metals in subjects with rhinitis. Methods. Patients suffering from rhinitis underwent a standard diagnostic procedure, including skin testing, nasal endoscopy and nasal cytology. Control healthy subjects were also included. None of the patients had skin diseases. All subjects underwent patch test with Chromium, Cobalt and Nickel. Results. None of the 26 controls had positive skin prick test or nasal cytology. The 82 rhinitis patients were subdivided into allergic (group A = 27), nonallergic (group B = 31) and overlapping (group C = 24). The prevalence of positive patch test to metals was 26% in group A, 45% in group B, 42% in group C and 31% in controls. The percentage of patch-positive subjects was significantly different between Group A and B (p = 0.0045; OR: 0.43), Group A and C (p = 0.0186; OR: 0.49), and Group B and controls (p = 0.0360; OR: 1.85). There was a significant difference between groups A + controls and B + C. Conclusion. Even in the absence of skin diseases, the prevalence of sensitization to metals (patch test) is greater in nonallergic and overlapping rhinitis, as compared to allergic rhinitis and controls.

Identification of a new small bioactive peptide from Lactobacillus gasseri supernatant.

Ten lactic acid bacteria (LAB) strains, previously isolated from human ileal biopsy of healthy volunteers, were screened for production and secretion of molecules having anti-bacterial and anti-biofilm activities. Because many recent reports indicate that LAB secreted molecules may exert immune-modulatory action, we also tested the effect on human intestinal HCT116 cells challenged with bacterial lipopolysaccharides. One of the Lactobacillus gasseri strains, SF1109, strongly inhibited: (1) Pseudomonas aeruginosa growth; (2) Escherichia coli biofilm production; (3) LPS induction of P-ERK1/2 in HCT116 cells, and was selected for further characterisation of the secreted active molecule. Cell-free supernatant of the L. gasseri SF1109 was analysed and one 1.3 kDa peptide has been characterised. Eight out twelve amino acids of this peptide were identified allowing the synthesis of an octa-peptide which still presented the mentioned activities.

Differentiation of human neuroblastoma cells toward the osteogenic lineage by mTOR inhibitor.

Current hypothesis suggest that tumors can originate from adult cells after a process of 'reprogramming' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic 'Warburg' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that 'in vitro' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate 'stem' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate.

Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm.

BACKGROUND: The use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases. Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown. METHODS: Spep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system. Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses. The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method. RESULTS: In this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity. CONCLUSIONS: New knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential "antipathogenic agent" capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity.

Antioxidant and antiapoptotic properties of melatonin restore intestinal calcium absorption altered by menadione.

The intestinal Ca²âº absorption is inhibited by menadione (MEN) through oxidative stress and apoptosis. The aim of this study was to elucidate whether the antioxidant and antiapoptotic properties of melatonin (MEL) could protect the gut against the oxidant MEN. For this purpose, 4-week-old chicks were divided into four groups: (1) controls, (2) treated i.p. with MEN (2.5 µmol/kg of b.w.), (3) treated i.p. with MEL (10 mg/kg of b.w.), and (4) treated with 10 mg MEL/kg of b.w after 2.5 µmol MEN/kg of b.w. Oxidative stress was assessed by determination of glutathione (GSH) and protein carbonyl contents as well as antioxidant enzyme activities. Apoptosis was assayed by the TUNEL technique, protein expression, and activity of caspase 3. The data show that MEL restores the intestinal Ca²âº absorption altered by MEN. In addition, MEL reversed the effects caused by MEN such as decrease in GSH levels, increase in the carbonyl content, alteration in mitochondrial membrane permeability, and enhancement of superoxide dismutase and catalase activities. Apoptosis triggered by MEN in the intestinal cells was arrested by MEL, as indicated by normalization of the mitochondrial membrane permeability, caspase 3 activity, and DNA fragmentation. In conclusion, MEL reverses the inhibition of intestinal Ca²âº absorption produced by MEN counteracting oxidative stress and apoptosis. These findings suggest that MEL could be a potential drug of choice for the reversal of impaired intestinal Ca²âº absorption in certain gut disorders that occur with oxidative stress and apoptosis.

New perspectives in melatonin uses.

This review summarizes the metabolism, secretion, regulation and sites of action of melatonin. An updated description of the melatonin receptors, including their signal transduction mechanisms, distribution and characterization of receptor genes, is given. Special emphasis is focused on the clinical aspects and potential uses of melatonin in the sleep-wake rhythms, in the immune function, in cancer therapy, in neuroprotection against oxidative damage and antioxidant activities in different tissues. Finally, combined effects of melatonin with other drugs are discussed.

A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium which possesses a variety of virulence factors, including many toxins, and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential anti-infective agent capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.

Proteomics analysis for the identification of three species of Thunnus.

The genus Thunnus comprises many species, some of higher quality and commercial value for their excellent organoleptic features, while others of lower quality and value. Consequently, these species are subjected to frequent fraudulent substitution. Increasing trade in fillet and minced fish makes the identification of external anatomical and morphological features of fish impossible. Proteomics was used for the identification of three Thunnus species. Muscle extracts were evaluated by both mono- and two-dimensional electrophoresis and mass spectrometric techniques. Preliminary results demonstrate that the tested species displays a high degree of polymorphism, making possible an accurate identification.

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing.

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotin-conjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed.

Pup circadian rhythm entrainment--effect of maternal ganglionectomy or pinealectomy.

In rodents, during late embryonic and early neonatal development, circadian rhythms develop in synchrony with those of their mothers, which in turn are synchronized with the environmental photoperiod. This paper examines the effect of maternal ganglionectomy (pineal gland sympathetic denervation) or extirpation of the pineal gland on pups' drinking rhythms, a behavior that is continuously monitored in individual animals starting after weaning and studied up to 3 weeks later. Maternal ganglionectomy or pinealectomy performed on the 7th day of gestation significantly disrupts rat pups' drinking behavior, within and among litters. In both treatments, circadian rhythm characteristics of the free-running period (tau), phase, amplitude and alpha were significantly altered compared to those of the control pups born from sham-operated mothers. With the exception of the alpha component, both maternal treatments have similar effects. When melatonin was given to the mothers instead of the endogenous pineal secretory activity for 5 days during the late period of gestation, this treatment reversed the effects of maternal ganglionectomy and pinealectomy. These observations, together with previous studies of our group, indicate that the maternal superior cervical ganglia and pineal gland are necessary components of the mechanism for maternal synchronization, and that maternal melatonin may, directly or indirectly, affect the performance of the pups' central oscillator during early pup rat development.

Indole-3-acetic acid improves Escherichia coli's defences to stress.

Indole-3-acetic acid (IAA) is a ubiquitous molecule playing regulatory roles in many living organisms. To elucidate the physiological changes induced by IAA treatment, we used Escherichia coli K-12 as a model system. By microarray analysis we found that 16 genes showed an altered expression level in IAA-treated cells. One-third of these genes encode cell envelope components, or proteins involved in bacterial adaptation to unfavourable environmental conditions. We thus investigated the effect of IAA treatment on some of the structural components of the envelope that may be involved in cellular response to stresses. This showed that IAA-treated cells had increased the production of trehalose, lipopolysaccharide (LPS), exopolysaccharide (EPS) and biofilm. We demonstrated further that IAA triggers an increased tolerance to several stress conditions (heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative stress) and different toxic compounds (antibiotics, detergents and dyes) and this correlates with higher levels of the heat shock protein DnaK. We suggest that IAA triggers an increased level of alert and protection against external adverse conditions by coordinately enhancing different cellular defence systems.

Separation and measurement of flame-formed high molecular weight polycyclic aromatic hydrocarbons by size-exclusion chromatography and laser desorption/ionization time-of-flight mass spectrometry.

The partial contribution of polycyclic aromatic hydrocarbons (PAH), capable of being detected by gas chromatography (GC-PAH), both to the total mass of the extractable organic fraction of flame-formed carbon particulates and to its UV-visible absorption and fluorescence spectra, has been determined by previous work. This contribution indicates the presence of PAH of molecular weight (MW) greater than 400 Da not accessible to conventional analysis. The detection of species in this higher MW range is important for both their potential toxicology and their possible role in soot formation. In the present work extracts of soots have been analyzed by linear mode laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) to extend the MW range that can be analyzed beyond the GC-PAH. The results have been compared with both analysis by reflector mode LDI-TOF-MS and the MW evaluation obtained by SEC analysis, as the shortcomings and advantages of both techniques appear to be complementary. Matching the results from the two techniques could give interesting insights in the molecular mass range between GC-PAH and the first soot particles (of mass > 2000 Da). Mass spectra in this molecular mass range have been obtained with a main ion sequence spacing of 24 Th and a minor ion sequence also with a spacing of 24 Th but off-set by 12 Th with respect to the main sequence. The two ion progressions have been interpreted by attributing the predominant peaks mainly to PAH with even-carbon numbers and the smaller ones to cyclopenta-fused ring PAH. These distributions indicate the occurrence of two competitive mechanisms in the growth of PAH and soot nucleation, i.e. the addition of acetylene (HACA mechanism) and the incorporation of pentagons by large polycyclic aromatic molecules into their aromatic bonding network.

Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: daily variations in phospholipid-synthesizing enzyme activities.

Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light-dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P-labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.

Long hours and little sleep: work schedules of residents in obstetrics and gynecology.

OBJECTIVE: To investigate residents' work schedules and their attitudes toward limiting their hours. METHODS: An anonymous survey regarding resident work hours and call schedules was administered to the 4674 obstetric-gynecologic residents who took the year 2000 Council on Resident Education in Obstetrics and Gynecology in-training examination. RESULTS: A total of 4510 surveys were analyzed (96.5%). Three of four (75.5%) respondents reported working between 61 and 100 hours each week. Most (71.3%) reported sleeping less than 3 hours while on night call. Eight of ten reported having postcall clinical responsibilities. The reported number of hours on call declined and the reported number of hours of sleep increased with year of residency. Three of four residents wanted limits on their work hours. Residents who reported longer on-call hours or less sleep during night shift were significantly more likely to want a restriction on work hours. Fatigue was the most commonly selected reason (77.6%) followed by "need more personal time" (76.3%), and "fear of compromising quality of care" (59.8%). Women were more concerned about fatigue than were men. Among residents who did not want work hour restrictions, "additional surgical experience" was the most commonly selected reason (69.0%). CONCLUSION: Residents in obstetrics and gynecology report working long hours, and experiencing periods of little sleep. Most want their work hours to be limited. Fatigue is a major concern among residents that want their hours limited. A sizable minority worries that such limits might also limit their experience.

The in-training examination in obstetrics and gynecology: an attempt to establish a remediation indicator.

OBJECTIVE: With the use of a university- and community hospital-based faculty, we attempted to determine at what performance level remediation would be recommended. STUDY DESIGN: The Committee on In-Training Examinations for Residents in Obstetrics and Gynecology Task Force on Standard-Based Scoring sent the 1991 examination to 16 university- and 12 community hospital-based faculty members. Given a standardized definition of a "borderline third-year resident," each faculty scored each item on the examination on whether that hypothetic resident would or would not correctly answer the item. RESULTS: The mean expectation of correct responses on the 397-item test was 236 (59%). This was identical to the score obtained if 2 SDs were subtracted from the actual mean for all third-year residents taking the examination. University- and community hospital-based faculty members had generally similar expectations of this defined resident. CONCLUSION: Although poor examination results should not be recommended as the sole determinant for promotion, it appears that 2 SDs below the mean may be an appropriate score below which remediation could be recommended.

Maternal coordination of the daily rhythm of malate dehydrogenase activity in testes from young rats: effect of maternal sympathetic denervation of the pineal gland and administration of melatonin.

Chronic sympathetic denervation of the pineal gland by bilateral removal of the superior cervical ganglia (SCG) was performed on female rats 30 days before impregnation. The offspring, maintained in the dark from birth, had disruption of the malate dehydrogenase circadian rhythm in the testes at 25 days of age. A daily injection of melatonin (1 mg/kg s.c. at 10:00 or 18:00 h) to denervated mothers from the 14th day of pregnancy up to the 10th day postpartum produced one daily phase in the enzyme activity of tests in the offspring. Entrainment of daily enzyme activity also was obtained when the hormone was administered orally to the pups during the postnatal period or when pups were reared by intact (not denervated) foster mothers. The results indicate the involvement of the maternal pineal gland in the maternal transfer of photoperiodic information necessary for the coordination of the circadian system in young rats.

Assessment of the resident in-training examination in obstetrics and gynecology.

An integral component for the evaluation of resident's cognitive knowledge is the examination developed under the auspices of the Council on Resident Education in Obstetrics and Gynecology (CREOG). We sought to assess the usefulness of this annual examination from the perspective of both residents and residency program directors. We were particularly interested in comparing the contemporary use of this examination with the original intent of CREOG when the examination was developed in 1968. In addition, we were interested in determining the role of the examination in modifying educational programs. A questionnaire was mailed to all program directors before the 1994 examination and given to all house staff when the examination was administered. The response rate was 55 and 82%, respectively. Overall, the majority of residents (60%) and program directors (58%) found the examination to be an accurate assessment of cognitive knowledge; feedback on examination results varied widely, and residents used a variety of tools to prepare for the examination.

Effect of sympathetic denervation of the pineal gland on maternal co-ordination of the circadian rhythm of alpha-amylase in parotid gland from young rats.

Twenty-five-day-old rats maintained in constant darkness since birth and born from mothers kept in the dark since the 14th day of pregnancy showed a circadian rhythm of alpha-amylase content in parotid glands, which may be explained by a mechanism of maternal co-ordination. Rats in the same conditions, except that their mothers had been submitted to bilateral excision of the superior cervical ganglia 30 days before mating, did not show diurnal variations of alpha-amylase activity in the parotid glands. When ganglionectomized mothers were treated with a daily dose of melatonin (1 mg/kg) from the 14th day of gestation up to the 10th day of lactation, their litters showed significant diurnal variations of amylase in the parotid glands, suggesting a role of the maternal pineal gland in the maternal-fetal and/or maternal-neonatal transfer of photoperiodic information.