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SUMMARYRNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme’s reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

Vitamins B (thiamine) and B (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5?-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5?-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg but activating the Zn-dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B- and B-dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in K of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased K of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness. (Figure presented.).

The role of aflatoxins (AFs) in the biology of producing strains, Aspergillus sect. Flavi, is still a matter of debate. Over recent years, research has pointed to how environmental factors altering the redox balance in the fungal cell can switch on the synthesis of AF. Notably, it has been known for decades that oxidants promote AF synthesis. More recent evidence has indicated that AF synthesis is controlled at the transcriptional level: reactive species that accumulate in fungal cells in the stationary growth phase modulate the expression of aflR, the main regulator of AF synthesis—through the oxidative stress related transcription factor AP-1. Thus, AFs are largely synthesized and secreted when (i) the fungus has exploited most nutritional resources; (ii) the hyphal density is high; and (iii) reactive species are abundant in the environment. In this study, we show that AFs efficiently scavenge peroxides and extend the lifespan of E. coli grown under oxidative stress conditions. We hypothesize a novel role for AF as an antioxidant and suggest its biological purpose is to extend the lifespan of AFs-producing strains of Aspergillus sect. Flavi under highly oxidizing conditions such as when substrate resources are depleted, or within a host.

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental problems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, “green”, approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no significant effect on mycotoxin inhibition; heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins.

The use of plant extracts in pre- and post-harvest disease management of agricultural crops to cope with aflatoxin B1 contamination has shown great promise due to their capability in managing toxins and safe-keeping the quality. We investigated the anti-aflatoxigenic effect of multiple doses of eight plant extracts (Heracleum persicum, Peganum harmala, Crocus sativus, Trachyspermum ammi, Rosmarinus officinalis, Anethum graveolens, Berberis vulgaris, Berberis thunbergii) on Aspergillus flavus via LC-MS and the down-regulatory effect of them on aflR, aflM and aflP genes involved in the aflatoxin B1 biosynthesis pathway using RT-qPCR analyses. Our results showed that H. persicum (4 mg/mL), P. harmala (6 mg/mL) and T. ammi (2 mg/mL) completely stopped the production of aflatoxin B1, without inducing significant changes in A. flavus growth. Furthermore, our findings showed a highly significant correlation between the gene expression and the aflatoxin B1 biosynthesis, such that certain doses of the extracts reduced or blocked the expression of the aflR, aflM and aflP and consequently reduced the synthesis of aflatoxin B1. Interestingly, compared to the regulatory gene (aflR), the down-regulation of expression in the structural genes (aflM and aflP) was more consistent and correlated with the inhibition of aflatoxin B1 production. Overall, this study reveals the anti-aflatoxigenic mechanisms of the selected plant extracts at the gene expression level and provides evidence for their use in plant and crop protection.

In recent years, algorithm research in the area of recommender systems has shifted from matrix factorization techniques and their latent factor models to neural approaches. However, given the proven power of latent factor models, some newer neural approaches incorporate them within more complex network architectures. One specific idea, recently put forward by several researchers, is to consider potential correlations between the latent factors, i.e., embeddings, by applying convolutions over the user-item interaction map. However, contrary to what is claimed in these articles, such interaction maps do not share the properties of images where Convolutional Neural Networks (CNNs) are particularly useful. In this work, we show through analytical considerations and empirical evaluations that the claimed gains reported in the literature cannot be attributed to the ability of CNNs to model embedding correlations, as argued in the original papers. Moreover, additional performance evaluations show that all of the examined recent CNN-based models are outperformed by existing non-neural machine learning techniques or traditional nearest-neighbor approaches. On a more general level, our work points to major methodological issues in recommender systems research., Comment: Source code available here: https://github.com/MaurizioFD/RecSys2019_DeepLearning_Evaluation

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental problems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, “green”, approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no significant effect on mycotoxin inhibition, heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins.

In this paper we provide an overview of the approach we used as team PoliCloud8 for the ACM RecSys Challenge 2019. The competition, organized by Trivago, focuses on the problem of session-based and context-aware accommodation recommendation in a travel domain. The goal is to suggest suitable accommodations fitting the needs of the traveller to maximise the chance of a redirect (click-out) to a booking site, relying on explicit and implicit user signals within a session (clicks, search refinement, filter usage) to detect the users intent. Our team proposes a solution based on several new features, designed to capture specific types of information as well as some well-known models: gradient boosting, neural networks and a stacking-based ensemble.

Background The Satellite Pharmacy analyses the organisation, processes, information flows and logistics related to the management of materials, mainly optimising the preparation of the procedural kits, the replenishment of the stock of medical devices (MD) at the storage locations of the surgery block operators, and returns management. By the very advanced radio frequency identification (RFID) technology according to which the products are equipped with a label containing the information of the product, we can trace the MDs from acceptance in the pharmacy to deposit in the RFID basket for surgical intervention and the patient, computerising the management procedures, rationalising the inventory management of the devices, and managing the procurement and purchasing processes with a minimum activity requirement by the operators. Purpose Improve clinical practice in the healthcare system by RFID technology, which shows efficiency and ability to manage a rational use of human resources and materials. Material and methods With the aim of providing some indicators that show a summary of information on the success of the activities of all the operators involved, we analysed: Allocated index: ratio between specific cost centre (CC) allocations vs. generic CC allocations; and Employment index: ratio between the total number of interventions performed and those that resulted without registering basket consumption. The monitoring of these parameters makes it possible to check the progress of the improvement objectives. Results The analyses of the cost (by intervention) recorded for the first 9 months of 2017 and 2018 in the same range of surgical specialists, shows an increase from 34% in 2018 of the total report, of which the amount allocated directly to the patient rose from 78.5% to 85.3%. In addition, in 2018 there was a reduction of 80% consumption recorded in intervention compared to 2017, of which the overall percentage of operations without associated MD dropped from 3.4% (2017) to 0.7% (2018). Conclusion The analysis and reports, processing through the collaboration between the various professions, has allowed a constant control of consumption and costs for each intervention, per patient and cost centre/operating room, ensuring better management of reporting flows at cost. References and/or acknowledgements No conflict of interest.

Celery is a widely used vegetable known for its peculiar sensorial and nutritional properties. Here, the white celery (Apium graveolens L.) "sedano bianco di Sperlonga" PGI ecotype was investigated to obtain the metabolic profile of its edible parts (blade leaves and petioles) also related to quality, freshness and biological properties. A multi-methodological approach, including NMR, MS, HPLC-PDA, GC-MS and spectrophotometric analyses, was proposed to analyse celery extracts. Sugars, polyalcohols, amino acids, organic acids, phenols, sterols, fatty acids, phthalides, chlorophylls, tannins and flavonoids were detected in different concentrations in blade leaf and petiole extracts, indicating celery parts as nutraceutical sources. The presence of some phenols in celery extracts was here reported for the first time. Low contents of biogenic amines and mycotoxins confirmed celery quality and freshness. Regarding the biological properties, ethanolic celery extracts inhibited the oxidative-mediated DNA damage induced by tert-butylhydroperoxide and scavenged DPPH and ABTS radicals.

In this work, the first phytochemical study on the total polar fraction of Linaria purpurea (L.) Mill. was performed by means of column chromatography and NMR and MS analysis. Seven compounds were identified i.e. pheophytin a (1), methyl-pheophorbide a (2), linaride (3), antirrhide (4), antirrhinoside (5), linarioside (6) and shikimic acid (7), belonging to three different classes of natural compounds. Compound (2) represents a new compound for the family as well as compound (1) is for the genus and compounds (3, 6, 7) are for the species. The chemosystematic and ethnopharmacological relevance of these results were widely discussed. In addition, compound (5) was tested, for the first time, for its potential antifungal activity, showing to drastically reduce the production of aflatoxin B1 in Aspergillus flavus Link. This further property of compound (5) makes it a potential natural and green anti-aflatoxin B1 agent to be used in the food industry.

A multi-methodological approach was applied to study red sweet peppers (Capsicum annuum L.) ecotype “Cornetto di Pontecorvo” grown in a greenhouse or in open field. This approach includes morphological analysis, chemical composition determination, and biological activity evaluation of different extracts from pepper fruits. Untargeted analyses, namely NMR spectroscopy and mass spectrometry, allowed the comprehensive pepper metabolite profile of pepper pulp, peel and seeds hydroalcoholic and organic extracts to be determined, showing the presence of sugars, organic acids, amino acids and other secondary metabolites. Targeted analyses, such as HPLC-PDA, HPLC-TLC and spectrophotometric analyses allowed polyphenols, tannins, flavonoids and pigments content to be determined. Samples quality and freshness were verified by the low content of biogenic amines and mycotoxins, as determined using HPLC-FLD and HPLC-MS, respectively. Preliminary biological results demonstrated the ability of the organic extracts to inhibit α-amylase, a key enzyme in the control of glucose metabolism.

Aflatoxins are bioactive compounds, which are toxic and carcinogenic for humans and animals, and are produced mainly by Aspergillus flavus and A. parasiticus. We studied the effect of some bioactive compounds produced into the culture filtrates of the basidiomycete Trametes versicolor on A. flavus growth and aflatoxin synthesis. Some aspects of the mechanism of action of different filtrate fractions were investigated.The results showed that these compounds stimulate the antioxidant system of the Aspergillus mycelia by the induction of a transient and modulated oxidative burst (peroxides and superoxide anions formation) perceived by Afyap1 transcription factor. T. versicolor culture filtrate fractions, significantly inhibited aflatoxin biosynthesis (A: 75% and B: 55%) apparently by stimulating endogenous antioxidant system. In conclusion, bioactive compounds present in the T. versicolor filtrates probably act by modulating the fungal antioxidant system, eventually leading to aflatoxin inhibition. T. versic...

Mushrooms represent a formidable source of bioactive compounds. Some of these may be considered as biological response modifiers ; these include compounds with a specific biological function: antibiotics (e.g. plectasin), immune system stimulator (e, g, lentinan), antitumor agents (e.g. krestin, PSK) and hypolipidemic agents (e.g. lovastatin) inter alia. In this study, we focused on the Chinese medicinal mushroom "yun zhi", Trametes versicolor, traditionally used for (cit.) "replenish essence and qi (vital energy)". Previous studies indicated the potential activity of extracts from culture filtrate of asexual mycelia of T. versicolor in controlling the growth and secondary metabolism (e.g. mycotoxins) of plant pathogenic fungi. The quest of active principles produced by T. versicolor, allowed us characterising an exo- polysaccharide released in its culture filtrate and naming it Tramesan. Herein we evaluate the biological activity of Tramesan in different organisms: plants, mammals and plant pathogenic fungi. We suggest that the bioactivity of Tramesan relies mostly on its ability to act as pro antioxidant molecule regardless the biological system on which it was applied.

A fresh analysis of this famous speech strengthens the idea that Cicero was unable to produce decisive proofs supporting the defendant. His arguments are in fact rather weak and often contradictory, so that some modern scholars have raised doubts about Roscius' innocence. However, there are not enough reasons to think so. Indeed Cicero could not get to the heart of this affaire, probably in consequence of the gloomy political situation, but Roscius was almost certainly not guilty. In spite of his faulty defensive strategy Cicero won this case and was entirely successful by virtue of the vehemence of his oratory. This paper aims at dispelling some doubts about the course of events, but much more remains obscure. Una rilettura di questa celebre orazione rafforza l'impressione che Cicerone non fu in grado di produrre prove schiaccianti a favore dell'accusato. La sua argomentazione é piuttosto debole e spesso contraddittoria, tanto che alcuni studiosi moderni hanno messo in dubbio l'innocenza di Roscio. Comunque non ci sono fondati motivi per crederlo. In realtá Cicerone non riuscí ad andare a fondo in questo affaire probabilmente a causa della torbida situazione politica, ma Roscio quasi certamente non fu colpevole. Nonostante la debolezza della sua strategia difensiva Cicerone vinse la causa e riscosse uno straordinario successo grazie alla veemenza della sua oratoria. Il presente articolo tende a dissipare qualche dubbio relativo allo svolgimento dei fatti, ma molto di piú é ció che resta avvolto nell'oscuritá.

Sénèque, De la clémence. Texte établi et traduit par François-Régis Chaumartin. Nouvelle édition. Paris: Belles lettres 2005. XCII, 125 S. z.T. Doppels. (Collection des Universités de France.) 31 €.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

An infant with normal facies and none of the extracardiac anomalies usually associated with Williams syndrome presented at birth with an echocardiographic pattern of supravalvular pulmonary stenosis and displastic pulmonary valve. A clinical reappraisal was planned at 3 months of age, but the girl died suddenly at home at 2 months of age. At autopsy, both ventricles were hypertrophic, and the valves showed mild dysplasia. The walls of the great arteries were thick, with a “washed leather” consistency, but there was no gross evidence of discrete stenosis. The histologic mosaic appearance of the media of the great arteries, due to elastosis and extreme disarray of the elastic lamellae, prompted a postmortem diagnosis of supravalvar aortic stenosis and suggested a diagnosis of Williams syndrome, which was subsequently confirmed by fluorescence in situ hybridization. Pediatricians and pathologists should be alerted that Williams syndrome in the newborn may present as an isolated supravalvular pulmonary stenosis, whereas supravalvular aortic stenosis becomes clinically significant only a few months later.

Suicide by simultaneous gunshots with two firearms is rare. The case of a 90-year-old man who killed himself with two 6.35 mm pistols fired at the temples at the same time is presented. The victim was found dead on the terrace of his home; two guns and two cartridges were present near the corpse. Cases of suicides with two guns published in the literature are compared, and the Italian cases are illustrated briefly. The authors describe the circumstances and autopsy findings that permitted the assertion that it was a case of suicide and to exclude an homicide and show, by figures, the three-dimensional reconstruction of cranial shooting injuries. The use of two guns is considered a representation of a special type of "combined suicide" or "planned complex suicide."

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is observed as a minor contaminant in caramel food colourings (E 150c). Feeding experiments with rodents have revealed a significant lymphopenic effect that has been linked to the presence of THI in these food colourings. Pyridoxal kinase inhibition by THI has been suggested, but not demonstrated, as a mode of action as it leads to lowered levels of pyridoxal-5'-phosphate, which are known to cause lymphopenia. Recently, THI was also shown to inhibit sphingosine-1-phosphate lyase causing comparable immunosuppressive effects and derivatives of THI are being developed for the treatment of rheumatoid arthritis in humans. Interestingly, sphingosine-1-phosphate lyase activity depends on pyridoxal-5'-phosphate, which in turn is provided by pyridoxal kinase. This report shows that THI does inhibit pyridoxal kinase with competitive and mixed-type non-competitive behaviour towards its two substrates, pyridoxal and ATP, respectively. The corresponding inhibition constants are in the low millimolar range.

The 34 cases of suicide by self-immolation admitted to the Institute of Forensic Medicine, University of Rome 'La Sapienza', during a 50-year period (1947-1997) were investigated. The nature of this phenomenon in relation to sex, age of the victims, location of the suicides and combination of methods used is discussed. Finally the distribution and extent and characteristics of the lesions are considered from a forensic point of view.

Our aim was to evaluate whether increased enterocyte apoptosis was responsible for mucosal flattening in celiac disease (CD), and, since the mechanisms responsible for tissue injury in this condition are unknown, we studied the possibility that the Fas-Fas ligand (FasL) system may be involved. Endoscopic duodenal biopsy specimens from 12 patients with untreated and 12 with treated CD and 12 control subjects were evaluated for enterocyte apoptosis by the terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine triphosphate nick-end labeling assay and for Fas and FasL expression by immunohistochemistry. A coculture of isolated enterocytes (targets) and purified lamina propria mononuclear cells (LPMCs) (effectors) was performed in the absence or presence of an antagonistic ZB4 antiFas antibody. We found a significant correlation between the degree of villous atrophy, morphometrically evaluated, and the level of enterocyte apoptosis, suggesting that mucosal flattening is a consequence of exaggerated epithelial cell death. Most celiac enterocytes express Fas, and LPMCs express FasL. The abolishment of enterocyte apoptosis observed in the presence of ZB4 antibody suggests that enterocytes are potential targets of lymphocyte infiltrate. These results directly demonstrate that FasLmediated apoptosis is a major mechanism responsible for enterocyte death in CD. Celiac disease (CD) is characterized by villous atrophy and crypt hyperplasia of the small intestinal mucosa caused, in genetically susceptible individuals, by a T-cell response to a new epitope generated by the transglutaminase-mediated

Potentiometric stripping analysis and constant current stripping analysis are proposed as routine methods for analysis of copper, zinc and selenium in plasma and urine samples. The analytical performance of these methods is comparable with that reported for atomic absorption spectrometry. However the low cost, greater simplicity of the apparatus, and the facility of execution make this methodology a valid candidate for routine application in Clinical Chemistry laboratories.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2–activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2–activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2–activated NK cells. Moreover, IL-2–induced interferon-γ production appears also to be dependent, at least in part, on iNOS induction.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2–activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2–activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2–activated NK cells. Moreover, IL-2–induced interferon-γ production appears also to be dependent, at least in part, on iNOS induction.

Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein–dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

The progression of HIV-1 disease appears associated with an unregulated Fas-mediated apoptosis of lymphocytes that involves the activation of ICE protease and ceramide generation and antiviral therapy may not be fully effective in the absence of a relevant impact on apoptosis. Six drug-naive HIV-1-infected symptomless patients with advanced immunodeficiency were treated with combined AZT and ddl for 4 months; plasma HIV-1 RNA levels, the counts of CD4 cells, CD4 and CD8 apoptotic lymphocytes, Fas-positive cells and ICE-positive cells, and intracellular ceramide levels were measured at base-line and after 7, 45 and 120 days of treatment. There was a prompt reduction in plasma viremia and a secondary increase in CD4 counts, but the treatment had no impact on apoptotic CD4 and CD8 lymphocytes, Fas-positive cells and ICE-positive cells, and on the intracellular levels of ceramide. A discrepancy exists between the positive impact of combined AZT and ddl treatment on plasma viral load and CD4 counts and the lack of any effect on the process of lymphocyte apoptosis. We suggest to use the measurement of apoptotic lymphocytes as a surrogate marker to predict, in combination with viral load and CD4 counts, a large proportion of the clinical effect of antiviral therapy.

Allozyme variation was used to investigate the genetic structure of the coral reef fishes Stegastes nigricans, Epinephelus merra and Acanthurus triostegus around New Caledonia. Each species was sampled from each of three sites in the ≃1000 km circumference of the lagoon of New Caledonia. Allelic variation was recorded for each species at 14, 13 and 17 loci, respectively, and heterozygosity diversity (Hs) was 0.082, 0.065 and 0.116, respectively. Analysis of genetic differentiation between sites produced inconsistent results between species, with spatial heterogeneity in two species (S. nigricans, Fst = 0.038; A. triostegus, Fst = 0.049) and homogeneity in one species (E. merra, Fst = 0.000). Hydrological and climatic data from the lagoon suggest that the eastern and western sides of the lagoon are isolated, since they lie in water masses of different origin. This may explain the genetic differentiation and restricted gene flow found at a local scale for S. nigricans and A. triostegus. Homogeneity in populations of E. merra is discussed in relation to its low genetic diversity and its reproductive behaviour.

SUMMARYSince in coeliac disease mucosal flattening has been suggested to result from an increased enterocyte apoptosis triggered by Fas/Fas ligand system and perforin cytolytic granules, we looked for a similar mechanism in autoimmune enteropathy. Moreover, we tried to assess whether enterocyte autoantibodies, which are the hallmark of autoimmune enteropathy, may be involved in triggering enterocyte apoptosis in this condition. Immunohistochemical staining with anti-Fas, -FasL and -perforin MoAb, and TUNEL technique were applied on endoscopic duodenal biopsies of two autoimmune enteropathy patients, two untreated coeliac patients and two biopsied controls. Cytotoxicity assays were carried out by incubating peripheral blood mononuclear cells from a healthy subject (effectors) with enterocytes primed with patient or control sera (targets). In autoimmune enteropathy a large number of enterocytes were apoptotic, as in coeliac disease, whereas neither Fas/Fas ligand or perforin expressions were up-regulated. On the other hand, antibody-dependent cellular cytotoxicity assay revealed the ability of sera from patients with autoimmune enteropathy to mediate enterocyte death through apoptosis. These results point to enterocyte autoantibody-dependent cellular cytotoxicity as the prevalent mechanism of increased enterocyte apoptosis in autoimmune enteropathy but not in coeliac disease.

This paper describes and evaluates a training program for parental educators to teach parenting classes for families with children 1 to 5 years of age. Six staff and parent volunteers from a family resource center participated. Evaluations of the training program showed improved knowledge and increased comfort in teaching parenting classes. Two graduates of the training program led a 10-week series of parenting classes. Parents in their class series ( n = 9) significantly reduced their reported use of verbal and corporal punishment, increased their nurturing, and improved their perceptions of their children's behavior. These results were similar to those from a series of similar classes for 15 parents led by a university instructor. Results are discussed in terms of research needs for training qualified parental educators.

The aim in this paper is to follow the evolution of this thread of classical geography from late antiquity through to the mixture of tradition and innovation which succeeded it in the medieval period, without any pretensions to being exhaustive in the naming of authors, and limiting the geographical region discussed to the north. ...

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2-activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2-activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2-activated NK cells. Moreover, IL-2-induced interferon-gamma production appears also to be dependent, at least in part, on iNOS induction.