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Kaposi sarcoma herpesvirus (KSHV), also known as human herpesvirus 8, is the causative agent of Kaposi sarcoma; this malignant angiosarcoma is usually treated with conventional antitumor agents that can control disease evolution, but do not clear the latent KSHV episome that binds to cellular DNA. Some commercial antibacterial sulfonamides were tested for the ability to suppress latent KSHV. Quantitative PCR (qPCR) and cytofluorometry assays were used for detecting both viral DNA and the latency factor LANA (latency-associated nuclear antigen) in BC3 cells, respectively. The capacity of sulfonamides to impair MDM2-p53 complex formation was detected by an enzyme-linked immunosorbent assay method. The analysis of variance was performed according to one-way analysis of variance with Fisher as a post hoc test. Here we show that sulfonamide antibiotics are able to suppress the KSHV latent state in permanently infected BC3 lymphoma cells and interfere with the formation of the MDM2-p53 complex that KSHV seemingly needs to support latency and to trigger tumor cell transformation. These findings detected a new molecular target for the activity of sulfonamides and offer a new potential perspective for treating KSHV-induced lymphoproliferative diseases.

Objective To investigate the link between Human Herpesvirus 8 (HHV8) infection and plasma oxidative stress in patients with diabetes mellitus type 2 (DM2). Results Blood samples collected from DM2 and control subjects were screened for the presence of antibodies against HHV8 and for biomarkers of oxidative stress. We determined the products of radical damage on the plasma lipid fraction, such as malondialdehyde (MDA), fatty acid hydroperoxides (HP) and 7-ketocholesterol (7-keto), the oxidation products of unsaturated fatty acids (UFA) and cholesterol, respectively. The level of plasma antioxidant α-tocopherol (α-toc) was also assessed. Relevant differences were observed in the redox status in DM2 and either HHV8-positive or -negative control subjects. The level of α-toc significantly decreased in both DM2 and HHV8-positive subjects. Levels of MDA, HP and 7-keto were much higher in HHV8-positive and DM2 subjects, indicating that plasma oxidative stress is a common feature in both DM2 and HHV8-infection. In addition, 7-keto was further increased in HHV8-positive DM2 patients. We hypothesized that the HHV8-infection may contribute to the production of ROS, and hence to the oxidative stress closely related to the pathogenesis and development of DM2.

Oncogenic and latent-persistent viruses belonging to both DNA (herpesviruses) and RNA viruses [hepatitis C virus (HCV), enteroviruses, rubella, human immunodeficiency virus, and dengue virus] are known to cause severe alterations of cell metabolism, which occasionally can lead to diabetes onset. HCV has been claimed to be able to impair cell metabolism by means of a direct modification of insulin cell sensitivity and host innate immune response. Human herpesvirus 8 (HHV8) infection also induces stable modifications in cell metabolism. HHV8 enhances the production of insulin receptors in endothelial cells, favors the concentration of lipid droplets in the cytoplasm of endothelial cells, and induces a general impairment of both triglycerides and cholesterol metabolism. HHV8 is also known to alter humoral response and the production of cytokines, which affects cell oxidative activity (reactive oxygen species activation), with a general impairment of acquired immunity. This review focuses particular attention on the recent findings reported on these two latent-persistent human viruses, namely, HCV and HHV8.

Oncogenic and latent-persistent viruses belonging to both DNA and RNA groups are known to cause serious metabolism alterations. Among these, the Human Herpesvirus 8 (HHV8) infection induces stable modifications in biochemistry and cellular metabolism, which in turn affect its own pathological properties. HHV8 enhances the expression of insulin receptors, supports the accumulation of neutral lipids in cytoplasmic lipid droplets and induces alterations in both triglycerides and cholesterol metabolism in endothelial cells. In addition, HHV8 is also known to modify immune response and cytokine production with implications for cell oxidative status (i.e., reactive oxygen species activation). This review underlines the recent findings regarding the role of latent and persistent HHV8 viral infection in host physiology and pathogenesis.

Introduction: Human Herpesvirus 8 (HHV8) is known to be the cause of the malignant tumour named Kaposi’s sarcoma. It is believed to induce an intense modification of cell metabolism in endothelial cells. In this work we analysed the role of anti-HHV8 antibodies in both the insulin and glucose uptake of HHV8-infected primary human endothelial cells (HUVEC). Methodology: Western blotting, immunofluorescence and radiolabelled glucose were employed to assess the pPI3K expression, insulin binding and glucose-uptake by HUVEC cells, respectively. Results: We confirmed that HHV8-infection is able to enhance both insulin binding and glucose-uptake in HHV8-infected primary endothelial cells; in addition, we found that anti-HHV8 specific antibodies are able to further increase both insulin and glucose uptake during the late latent phase of HHV8-infection in vitro. Conclusions: These findings suggest that a specific immune response to HHV8-infection may cooperate in boosting the cell metabolism, further enhancing the already increased insulin binding and glucose-uptake in HHV8-infected cells, which is a peculiar property of several oncogenic viruses.

The dichloromethane extract of the leaves of Bupleurum fruticosum was found to inhibit the replication of human rhinovirus (HRV) serotypes 14 and 39. Bioassay-guided fractionation led to the isolation of seven phenylpropenol derivatives (3–9), two polyacetylenes (1 and 2), and one monoterpene (10). Compounds 1 and 10 were identified as previously undescribed secondary metabolites after extensive 1D and 2D NMR experiments as well as high-resolution mass spectrometry. Compounds 2, 4, and 5 showed a selective inhibition of viral replication against HRV39 serotype, with 2 and 4 being the most active, with EC50 values of 1.8 ± 0.02 and 2.4 ± 0.04 μM. Mechanism of action studies indicated that 4 behaves not only as a capsid binder, interfering with the early phases of virus replication, but also as a late-phase replication inhibitor. Docking experiments were performed to confirm the ability of the antiviral phenylpropenoids to selectively fit into the hydrophobic pocket of VP1-HRV39.

A high prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological conditions of diabetes contribute to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells were cultured in high-glucose medium. To demonstrate the association between high glucose levels and KSHV replication, we xenografted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV cells) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose levels induced the production of hydrogen peroxide (H2O2), which downregulated silent information regulator 1 (SIRT1), a class III histone deacetylase (HDAC), resulting in the epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose levels in diabetic patients contribute to the development of KS by promoting KSHV lytic replication and infection.

The development of type 2 diabetes is thought to involve both environmental, possibly infectious, and genetic factors. Recently, a high prevalence of human herpesvirus 8 (HHV8) infection was observed in type 2 diabetes patients, and specific killer cell immunoglobulin-like receptors (KIR) allotypes were associated to both increased susceptibility to herpesvirus infection and risk to develop diabetes. However, no clear gene-disease or virus-disease associations have been established. To investigate the possible interplay between HHV8 infection, KIR allotype and type 2 diabetes, virus prevalence and KIR genotype were analyzed by PCR in 168 patients affected by type 2 diabetes and 108 control individuals belonging to the Sardinian population. Results showed a significant increase of HHV8 prevalence in type 2 diabetes patients versus controls (57% vs. 17%, P

The prevalence of Human Herpesvirus 8 (HHV8) DNA and antiviral antibodies in Diabetes type 2 (DM2) and control subjects was studied, in order to confirm a possible link between DM2 and HHV8 infection. The HHV8-DNA from diabetic patients was typed for detecting possible genomic differences with known HHV8 reference viruses.DM2 patients and healthy controls were examined for the presence of HHV8 DNA into the peripheral blood lymphocytes. Both anti-lytic and latent phase antibodies were detected in HHV8 positive and negative diabetic patients, as well in a number of controls. The HHV8 ORF K1 and ORF 26 genes from DM2 patients were typed and matched to reference strains.A significant prevalence of HHV8 DNA in DM2 subjects versus healthy controls was detected (about 58 % against 27 %). Anti-lytic phase, but not anti-latent phase antibodies, were significantly increased in DM2 patients versus controls. In addition, about 30 % of HHV8 strains isolated from DM2 lymphocytes showed consistent differences in the ORF 26 gene sequence, so that a new HHV8 subtype was proposed. These findings give additional support to the hypothesis that HHV8 could be considered an additional risk factor for DM2 onset.

The triterpene glycyrrhizic acid (GRA), the main product from the Glycyrrhiza glabra medicinal plant, is known for its antiinflammatory and antimicrobial activity. In this work, GRA was studied for its ability to induce the autophagic process activator Beclin 1 in epithelial cells and to observe how this property could influence its antiviral activity. After 24 h of treatment, GRA induced a Beclin 1 production that was more than twofold higher than that produced by rapamycin, used as a reference compound. When the compounds were added to HeLa cells together with the viruses, GRA demonstrated a strong antiherpes simplex virus type 1 (HSV1) activity, whereas rapamycin had no activity. However, if the compounds were added to the cells 24 h before the viruses, GRA induced the production of an even higher amount of Beclin 1 and showed an improved antiviral effect; under these conditions, rapamycin was also able to exert a significant anti-HSV1 activity. In conclusion, GRA is a strong inducer of the autophagy activator Beclin 1, which establishes a resistance state to HSV1 replication. Copyright © 2014 John Wiley & Sons, Ltd.

Three novel benzophenones (1–3) were obtained from the roots of Securidaca diversifolia (L.) S.F. Blake along with two known benzylbenzoates and isovanilline. Compound 2 (2,4-dihydroxy-3,3′-dimethoxy-benzophenone) showed moderate inhibitory activity on Herpes simplex virus type 1 proliferation in vitro. The structures of compounds 1–3 were established by the interpretation of their spectroscopic and spectrometric data.

Problem statement: The possible contribution of viruses to vascular pathology is still a controversial issue. Human herpesvirus type 8 (HHV-8) has been suggested to participate to the pathogenetic events associated with atherosclerotic lesion establishment and progression. Recently, a high incidence of infection of HHV-8 (11%) has been verified in the island of Sardinia. The aim of this study was to evaluate a possible relationship between the HHV-8 infection and cardiovascular diseases in the South of Sardinia. Approach: The presence of HHV-8 genome was detected in DNA extracted from peripheral leucocytes, by nested-PCR and Southern blotting, in either acute or chronic cardiopathic patients (n = 180); healthy blood donors were examined as controls (n = 108). Results: The results demonstrated a significant increase (p = 0.035) in HHV-8 DNA isolation from cardiopathic patients (22.8%) in comparison to healthy controls (12.0%). Conclusion: HHV8 infection can be considered, among others, as an additional risk factor for cardiovascular disease development, although it was not necessarily the starting cause. More extensive studies were needed to define the exact role of HHV-8 infection in cardiopathic patients.

Solid-state (13)C nuclear magnetic resonance (NMR) and elemental analysis techniques were used to monitor the degradation of wheat bran by the white-rot fungus Pleurotus ostreatus during a 62-day cultivation period. The weight loss and in vitro organic matter digestibility of the substrate were also evaluated after fungal treatment. The (13)C NMR spectra of degraded wheat bran samples showed a lower content in carbohydrates and a higher content in aliphatic and carboxylic groups than the untreated control sample. In parallel, changes in the wheat bran elemental composition evidenced a decrease in carbon content and a concomitant increase in nitrogen and oxygen content during mycelium growth. These results clearly indicate the occurrence of progressive changes in the composition of wheat bran during fungal treatment and are interpreted in terms of preferential degradation of amorphous vs. crystalline polysaccharides by the fungal mycelium and accumulation of proteins in the substrate. At the end of the cultivation period, the treated samples experienced an average weight loss of 20% and an increase in organic matter digestibility of 17%.

The prevalence of Human Herpesvirus 8 (HHV8) DNA and antiviral antibodies in Diabetes type 2 (DM2) and control subjects was studied, in order to confirm a possible link between DM2 and HHV8 infection. The HHV8-DNA from diabetic patients was typed for detecting possible genomic differences with known HHV8 reference viruses.

The isolation and identification of Listeria monocytogenes in processed meat samples by a combined cultural-molecular method is described. It allows the identification of Listeria strains by means of a hybridization technique with a specific DNA probe directed to the listerial internalin gene. The specificity of this method was found to be 100% and sensitivity was as low as 1 CFU/2.5 g of food sample. A total of 278 meat samples were tested in comparison with PCR and conventional cultural assays. A total of 42 (15.4%) L. monocytogenes were detected. PCR analysis gave 3 false negative results and culture failed to detect the Listeria in 5 cases. With this cultural-molecular method the identification and quantitative detection of L. monocytogenes were achieved within 36 hours and no false positive or negative tests were obtained, thus fitting most food industry requirements.

In this study a possible relationship between the presence of type 2 diabetes (DM2) and the Human Herpesvirus 8 infection (HHV-8) has been evaluated. The presence of HHV-8 DNA was monitored by nested PCR and Southern blotting in peripheral blood leucocytes of DM2 patients admitted to the Diabetology and Metabolic Disease Service of the &#34S. Giovanni di Dio” Hospital (Cagliari, Italy); healthy blood donor subjects were examined as controls. The results suggest that the frequency of HHV-8 DNA detection is more elevated in DM2 patients (23.7%) than in healthy control subjects (12%, p

58 patients with chronic osteomyelitis were treated either with a standard therapy or with a specific targeted therapy based on the antibiogram assay. Standard therapy was performed by a 15-day course of a parenteral cephalosporin (usually ceftriaxone) in combination with an aminoglycoside (e.g. netilmicin), followed by oral therapy with a fluoroquinolone (generally ciprofloxacin) for 1 to 3 months; specific therapy largely varied depending on the antibiogram response. The results indicated that no significant differences were found between the patients who received standard therapy (95.5 % cured) and those who received a specific therapy (93.5 % cured), after a one year follow-up. It can be concluded that a short-term combined parenteral-oral standard therapy, indicated in all those cases where antibiotic therapy must be started before obtaining the laboratory response, or when a clear and definite identification of the microorganisms involved in the infection is not possible, can be as efficient as an antibiogram-guided therapy in the treatment of chronic osteomyelitis.

Laboratory experiments were performed to characterize the behaviour of an electrochemical cell equipped with boron-doped diamond anodes and to verify its effectiveness in water disinfection. The hydrodynamic regime was determined when the cell worked either in batch or in continuous mode. Galvanostatic electrolyses of aqueous 1 mM Na2SO4 solutions were performed to investigate on the oxidant production in different experimental conditions. The same solutions contaminated by E. coli, enterococci and coliforms were used as test media to verify the effectiveness of the system in the disinfection process. Experimental results indicated that the major inactivation mechanism of bacteria in the electrochemical cell is a disinfection by electrochemically generated oxidants, however a cooperative effect of superficial reaction has to be taken into account. The great capability of BDD anode to produce reactive oxygen species (ROS) and other oxidizing species during the electrolysis allows to establish a chlorine-free disinfection process.

Diabetes is a common chronic disease due to an altered glucose metabolism, caused by the quantitative and/or qualitative dysfunction of the insulin hormone. Two types of diabetes are recognized: juvenile diabetes, or type 1, which has an autoimmune origin, and adult diabetes, or type 2 (DMT2), which covers 90-95 % of all diabetic patients.The causes of DMT2 are not yet clear: heredity, life style, nutrition, and environment are considered the main risk factors. Several viral infections, namely cytomegalovirus, coxsackie and other enteroviruses, rubella and hepatitis C virus, have been claimed to be associated with some forms of diabetes. The direct role of viruses as a cause or as a risk of type 1 diabetes has been amply described in several recent reviews. Therefore, this review focuses attention on the role of a human herpes pathogenic virus in the onset of DMT2. By carrying out an analysis of recent literature, we describe the findings reported on an extremely deceitful virus, such as Human Herpes virus 8, and present a medical hypothesis on a possible relationship between this virus and DMT2.

The bacterial species Streptococcus mutans is known as the main cause of dental caries in humans. Therefore, much effort has focused on preventing oral colonization by this strain or clearing it from oral tissues. The oral cavity is colonized by several bacterial species that constitute the commensal oral flora, but none of these is able to interfere with the cariogenic properties of S. mutans. This paper describes the interfering ability of some nutritionally variant streptococcal strains (NVS) with S. mutans adhesion to glass surfaces and also to hydroxylapatite. In mixed cultures, NVS induce a complete inhibition of S. mutans microcolony formation on cover glass slides. NVS can also block the adherence of radiolabeled S. mutans to hydroxylapatite in the presence of both saliva and sucrose. The analysis of the action mechanism of NVS demonstrated that NVS are more hydrophobic than S. mutans and adhere tightly to hard surfaces. In addition, a cell-free culture filtrate of NVS was also able to interfere with S. mutans adhesion to hydroxylapatite. Since NVS are known to secrete some important bacteriolytic enzymes, we conclude that NVS can be a natural antagonist to the cariogenic properties of S. mutans.

Background The Enterovirus genus of the Picornaviridae is represented by several viral pathogens that are associated with human disease, namely Poliovirus 1, Enterovirus 71 and Rhinoviruses. Enterovirus 71 has been associated with encephalitis, while Rhinoviruses are a major cause of asthma exacerbations and chronic obstructive pulmonary disease. Based on the structure of both pleconaril and pirodavir, we previously synthesized some original compounds as potential inhibitors of Rhinovirus replication. Methods These compounds were explored for in vitro antiviral potential on other human pathogenic Enteroviruses, namely Enterovirus 71 on rhabdo-myosarcoma cells, Coxsackievirus B3 on Vero cells, Poliovirus 1 and Echovirus 11 on BGM cells. Results Activity was confirmed for compound against Rhinovirus 14. Furthermore, few compounds showed a cell-protective effect on Enterovirus 71, presented a marked improvement as compared to the reference drug pleconaril for inhibitory activity on both Enterovirus 71 and Poliovirus 1. The most striking observation was the clear cell protective effect for the set of analogues in a virus-cell-based assay for Echovirus 11 with an effective concentration (EC50) as low as 0.3 µM (Selectivity index or SI = 483), and selectivity indexes greater than 857 (EC50 = 0.6 µM) and 1524 (EC50 = 0.33 µM). Conclusion Some of the evaluated compounds showed potent and selective antiviral activity against several enterovirus species, such as Enterovirus 71 (EV-A), Echovirus 11 (EV-B), and Poliovirus 1 (EV-C). This could be used as a starting point for the development of other pleconaril/pirodavir-like enterovirus inhibitors with broad-spectrum activity and improved effects as compared to the reference drugs.

On the basis of suggestions derived either from a pharmacophoric model for antitubercular agents or from a structure-activity relationship analysis of many pyrroles previously described by us, we report here the design and synthesis of new analogues of 1,5-(4-chlorophenyl)-2-methyl-3-(4-methylpiperazin-1-yl)methyl-1H-pyrrole (BM212). Various substituents with different substitution patterns were added to both positions 1 and 5 of the pyrrole nucleus to evaluate their influence on the activity toward Mycobacterium tuberculosis (MTB) and atypical mycobacteria. Biological data showed that, although some nontuberculosis mycobacterial strains were found to be sensitive, MIC values were higher than those found toward MTB. The best compound (1-(4-fluorophenyl)-2-methyl-3-(thiomorpholin-4-yl)methyl-5-(4-methylphenyl)-1H-pyrrole, 5) possessed a MIC of 0.4 microg/mL (better than BM212 and streptomycin) and a very high protection index (160), better than BM212, isoniazid, and streptomycin (6, 128, and 128, respectively). Finally, molecular modeling studies were performed to rationalize the activity of the new compounds in terms of both superposition onto a pharmacophoric model for antitubercular compounds and their hydrophobic character.

Background: Over the last decade several papers have dealt with the possible interference of allergies in both the infectious disease incidence and tumour development. In the light of all these observations we analysed several tumour patients for a possible interaction between a state of allergy and tumour development and progression after primary cancer therapy. Methods: This study included 1,055 patients with different types of solid tumours admitted consecutively between 1994 and 2002 to the Cagliari University Polyclinic. After primary surgery or medical therapy (or both), 92 allergic subjects and 182 non-allergic patients were studied over a follow-up period of 6–96 months (median 23). Results: Among 1,055 tumour-bearing patients, the prevalence of allergy was found to be about 8% versus 16–37% in a population of non-tumour-bearing subjects. After primary cancer therapy, allergic patients turned out to have a 20% higher probability of being cured and about a 50% lower risk of tumour progression as compared to non-allergic ones. The observed differences were statistically significant (p = 0.013). Conclusions: On the basis of our findings, we suggest that allergic subjects seem to have a better prognosis than non-allergic ones for disease outcome after cancer therapy.

During the course of our investigations in the field of azole antimicrobial agents, we have identified BM 212, a pyrrole derivative with good in vitro activity against mycobacteria and candidae. These findings prompted us to prepare new pyrrole derivatives 1-10 in the hope of increasing the activity. The microbiological data showed interesting in vitro activity against Mycobacterium tuberculosis and atypical mycobacteria.

Diabetes is a common chronic disease due to an altered glucose metabolism, caused by the quantitative and/or qualitative dysfunction of the insulin hormone. Two types of diabetes are recognized: juvenile diabetes, or type 1, which has an autoimmune origin, and adult diabetes, or type 2 (DMT2), which covers 90–95 % of all diabetic patients.

The present work was carried out to study the effect of some plant methanol extracts and essential oils on lipid peroxidation in simple in vitro systems. The tested extracts were obtained from four plants, commonly known in the Mediterranean area, indigenous to Sardinia: Artemisia arborescens L., Calycotome villosa L., Daphne gnidium L. or naturalized in the island, Eucalyptus globulus Labill. The activity of the extracts was investigated during both autoxidation and iron or EDTA-mediated oxidation of linoleic acid at 37 degrees C in the absence of solvent, and compared with that of BHT, alpha-tocopherol and EDTA. During linoleic acid autoxidation all the extracts were active, showing an antioxidant activity in the order: BHT >alpha- tocopherol >Daphne gnidium (methanol extract) >Eucalyptus globulus (essential oil) >Calycotome villosa (essential oil) >Artemisia arborescens (essential oil and methanol extract) >Calycotome villosa (methanol extract). None showed any prooxidant activity. During the iron-catalysed oxidation of linoleic acid the oils were not active, while all the methanol extracts showed some efficiency in preventing the oxidation process. All the extracts were also tested on cell cultures to investigate their cytotoxic activity or their ability to inhibit the growth of some pathogenic microorganisms.

The chemical composition of the essential oil and methanol extract of Calycotome villosa (Poiret) Link leaves collected in Sardinia (Italy) has been studied by analytical and spectroscopic methods. Falcarinol and some alcohols, terpenes, furan derivatives, and paraffins have been isolated from the essential oil. Thirteen alkaloids and falcarinol have been identified in the chloroform fraction of the basic methanol extract. Six flavonoids and four anthraquinones have been isolated in the chloroform fraction after acidification of the basic methanol extract. The cytotoxic and antimicrobial activities have also been evaluated. The essential oil, the methanol extract in toto, and the fraction of the basic extract showed strong cytotoxicity, whereas the fraction of the acid extract showed lower cytotoxicity. Furthermore, this fraction showed good antibacterial activity against Staphylococcus aureus, Bacillus lentus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Providencia rettgeri, and Morganella morganii. It can therefore be stated that this plant's cytotoxicity is prevalently due to falcarinol.

Some enzymes present in biological fluids, such as lysozyme (LYS) and lactoferrin (LAC), are known to possess antibacterial and antiviral activity, against herpesviruses in particular. It will be shown in this paper that their combination with a natural triterpene, namely glycyrrhizic acid (GLA), gives significant results in enhancing the antagonistic activity on HSV1 in in vitro assays. Data elaboration was carried out by calculation of the FIC index (fractional inhibitory concentration) for each combination of the three compounds and by a three-dimensional evaluation of the inhibiting combinatory effects, which indicated the percentage of the synergistic action. A FIC index equal to or below 0.5 demonstrated a significant synergistic effect between two substances. Considering each single compound, the 50% inhibiting doses on viral replication (ID50) were 252±53 μg/ml for LAC, 497±165 μg/ml for LYS and 740±125 μg/ml for GLA. The combination of LAC and GLA showed a clear synergistic effect, with a FIC index of 0.08 and a potentiating activity which, for some doses, was up to 1.5 log10 of difference (from about 5.5×106 to 105 pfu/ml). The combinations of GLA and LYS, and LYS and LAC showed a less significant synergistic activity. These findings led to the conclusion that some physiological proteins, even at concentrations usually present in some body fluids, may enhance the anti-herpetic activity of a natural compound such as GLA.

Santolina insularis was used to obtain its essential oil by means of supercritical fluid extraction with carbon dioxide, CO2. The choice of the proper value of CO2 density is the crucial point influencing solvent power and selectivity, the main factors determining oil composition. In circuit to obtain a pure essential oil deprived of cuticular waxes, the extraction products were fractionated in two separators operated in series. A good process performance was obtained working at 90 bar and 50 °C in the extraction vessel, at 90 bar and −12 °C in the first separator and at 20 bar and 15 °C in the second separator. The extraction time proved to be another parameter that determines the composition of the essential oil extracted. Indeed, lower molecular weight and less polar compounds were more readily extracted, since the other families of compounds exhibit higher diffusion times. The oil extracted also contained small quantities of water. The water yield was measured and the possible role of water in the extraction process is discussed. A comparison with the oil obtained by hydrodistillation is also given. Finally, the cytotoxic and antimicrobial activity of the various extracts has been assayed. Copyright © 2001 John Wiley & Sons, Ltd.

The antimicrobial activity of stems methanol extract from Daphne gnidium L. collected from Sardinia (Italy) was evaluated against 6 strains of standard and clinical isolated gram (+/-) bacteria. The antimicrobial effect on two strains of fungi was also tested. The extract in toto exhibited antibacterial activity against Bacillus lentus and Escherichia coli, but was inactive against fungi. Four coumarins (daphnetin, daphnin, acetylumbelliferone, daphnoretin) and seven flavonoids (luteolin, orientin, isoorientin, apigenin-7-O-glucoside, genkwanin, 5-O-beta-D-primeverosyl genkwanine, 2,5,7,4'-tetrahydroxyisoflavanol) present in the plant extract were also investigated against the same strains of bacteria and fungi assayed for the crude extract. The most active compounds were daphnetin, genkwanin, and 2,5,7,4'-tetrahydroxyisoflavanol.

Novel toluidine derivatives are described. Some of them showed an interesting in vitro activity against Mycobacterium tuberculosis, M. smegmatis, M. marinum, M. gordonae, and M. avium. Some of them were more active than Streptomycin and Isoniazid, which were used as controls, against M. avium and M. gordonae. In particular, we confirm the good activity of biphenyl derivatives.

Cheese whey and cottage cheese whey are by-products of the milk and cheese industry, resulting from the production of cheese and cottage cheese (ricotta) from milk. They are still rich in organic substances and cannot be discarded into the environment without proper treatment. Whey and cottage cheese whey were used as culture media for some strains of the yeast Kluyveromyces lactis, transformed with the human lysozyme gene. It was found that the yeast strains grew well in both media and produced a considerable amount of recombinant protein. Production kinetics showed that the human lysozyme was produced in a greater amount within 36 h of fermentation (125 micrograms ml-1 vs 25 micrograms ml-1 in the control) than in the synthetic commercial media used for strain preparation and characterization. The recombinant protein produced was actually shown to be the human lysozyme, using renaturing SDS-PAGE and Western blot techniques. While producing recombinant protein, the Kluyveromyces strain cleared the cottage cheese whey of most organic substances and produced a considerable amount (almost 3%) of lysozyme-enriched useful biomass.

Thiomorpholine analogues of U-100480 with the biphenylmethyl group replacing the acetamidomethyloxazolidinone moiety have been synthesized and tested as antimycobacterial agents together with various related derivatives. Some biphenyl derivatives were endowed with high activity against Mycobacterium tuberculosis and other non-tuberculous mycobacteria.

The human common cold, which is a benign disease caused by the Rhinoviruses, generally receives palliative symptomatic treatments, since no specific therapy against any of these viruses currently exists. In this work, some original synthetic compounds were produced and tested, in order to find non-toxic substances with an improved protection index (PI) for infected cells, as compared to reference drugs such as Pirodavir. We designed a series of novel molecules with a double oxygen in the central hydrocarbon chain and some modifications of the lateral methylisoxazole and propoxybenzoate moieties of lead compound 6602 (ethyl 4-{3-[2-(3-methyl-1,2-isoxazol-5-yl)ethoxy]propoxy}benzoate). It was found that most of these substances were actually less toxic than Pirodavir; in addition, the new molecule indicated as 8c was more than 30 times less toxic than Pirodavir, about twice as active on the group A strain of Rhinovirus HRV14, and even four times more effective on the group B strain HRV39, as compared to Pirodavir’s PI.

The triterpene glycyrrhizic acid (GRA), the main product from the Glycyrrhiza glabra medicinal plant, is known for its antiinflammatory and antimicrobial activity. In this work, GRA was studied for its ability to induce the autophagic process activator Beclin 1 in epithelial cells and to observe how this property could influence its antiviral activity. After 24 h of treatment, GRA induced a Beclin 1 production that was more than twofold higher than that produced by rapamycin, used as a reference compound. When the compounds were added to HeLa cells together with the viruses, GRA demonstrated a strong antiherpes simplex virus type 1 (HSV1) activity, whereas rapamycin had no activity. However, if the compounds were added to the cells 24 h before the viruses, GRA induced the production of an even higher amount of Beclin 1 and showed an improved antiviral effect; under these conditions, rapamycin was also able to exert a significant anti-HSV1 activity. In conclusion, GRA is a strong inducer of the autophagy activator Beclin 1, which establishes a resistance state to HSV1 replication.

In total 34 strains of Gardnerella vaginalis were analyzed with various molecular techniques in order to find the possibility of dividing this single species into different genotypes. Classical ribotyping, PCR-ribotyping and restriction analysis of 16S-23S rRNA intergenic spacer sequences were all unsuccessful in genotype differentiation of these bacteria. Only amplified ribosomal DNA restriction analysis (ARDRA) was suitable in recognizing different G. vaginalis genotypes. At least 3-4 genotypes were identified with different restriction enzymes, some of which showed a prevalent distribution in certain of the centers from which they were collected. Although in this study no correlation was found between bacterial vaginosis and any of the genotypes identified, the ARDRA method could prove to be a useful tool for studying the etiopathology and epidemiology of G. vaginalis.

Lasers are commonly used in several fields of medicine as a complementary therapy for internal medicine, surgery and also diagnostics. The efficacy of ultra-low level laser therapy (ULLLT) at power levels around 0.15 mW/cm(2) has been demonstrated both in in vitro experiments and in the clinical environment. This work used an ULLLT laser source to analyze its efficacy on Staphylococcus aureus adhesion to cells and on its ability to produce pathogenic factors. Laser stimulation succeeded in impairing the binding of S. aureus to primary human cells in culture and in inhibiting the expression of coagulase, one of the main staphylococcal pathogenic factors. The importance of the extracellular matrix (ECM) and the modification of the ECM redox potential in these activities were also evidenced.

A novel system for the culture and isolation of the micro-aerophilic Campylobacter species from foods and the environment is described. It consists of a plastic plate containing some linear wells filled with selective media, where a micro-aerophilic atmosphere can be developed. In this kit, called Linearcount 3MA, various Campylobacter species grew well, producing normal-size colonies in 48 h.

Internalin A (InlA), a protein required forListeria monocytogenesvirulence, is encoded by theinlA gene, which is only found in pathogenic strains of this genus. One of the best ways to detect and confirm the pathogenicity of the strain is the detection of one of the virulence factors produced by the microorganism. This paper focuses on the design of an electrochemical genosensor used to detect theinlA gene inListeriastrains without labelling the target DNA. The electrochemical sensor was obtained by immobilising aninlA gene probe (single-stranded oligonucleotide) on the surfaces of screen-printed gold electrodes (Au-SPEs) by means of a mercaptan-activated self-assembled monolayer (SAM). The hybridisation reaction occurring on the electrode surface was electrochemically transduced by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. The covalently immobilised single-stranded DNA was able to selectively hybridise to its complementary DNA sequences in solution to form double-stranded DNA on the gold surface. A significant decrease of the peak current of the voltammogram (DPV) upon hybridisation of immobilised ssDNA was recorded. Whole DNA samples ofL. monocytogenesstrains could be discriminated from other nonpathogenicListeriaspecies DNA with theinlA gene DNA probe genosensor.

Human Herpesvirus 8 (HHV8), the causative agent of Kaposi sarcoma, induces a profound modification of infected cell behaviour, with reprogramming of gene expression and changes in physiological properties, over-expression of the insulin receptor, increased resistance to stress conditions and prolonged cell survival in conditions of serum deprivation. This paper shows that HHV8 infection induces a strong enhancement of both insulin and glucose uptake in primary endothelial cells (HUVEC). The increase in insulin uptake is already evident in the lytic phase of the viral infectious cycle, and reaches a maximum of up to 71% during the latent phase, whilst glucose uptake is slightly depressed during the lytic viral infection, but significantly enhanced compared with the control during the latent phase of viral infection, with an average increase of about 37% 25 days after cell infection.

A highly active thermostable β-glucosidase was purified to homogeneity from a strain ofTrichoderma sp. The enzyme was an extracellular glycoprotein and showed hydrolytic activity toward several β-glucosides. Cellobiose was found to be the substrate of choice for this enzyme. This finding could suggest future technological applications of the purified protein.

Picornaviridae represent a very large family of small RNA viruses, some of which are the cause of important human and animal diseases. Since no specific therapy against any of these viruses currently exists, palliative symptomatic treatments are employed. The early steps of the picornavirus replicative cycle seem to be privileged targets for some antiviral compounds like disoxaril and pirodavir. Pirodavir’s main weakness is its cytotoxicity on cell cultures at relatively low doses. In this work some original synthetic compounds were tested, in order to find less toxic compounds with an improved protection index (PI) on infected cells. Using an amino group to substitute the oxygen atom in the central chain, such as that in the control molecule pirodavir, resulted in decreased activity against Rhinoviruses and Polioviruses. The presence of an -ethoxy-propoxy- group in the central chain (as in compound I-6602) resulted in decreased cell toxicity and in improved anti-Rhinovirus activity. This compound actually showed a PI >700 on HRV14, while pirodavir had a PI of 250. These results demonstrate that modification of pirodavir’s central hydrocarbon chain can lead to the production of novel derivatives with low cytotoxicity and improved PI against some strains of Rhinoviruses.

The phosphatase activities of cells of representative enterobacterial species were studied after cells were grown in a chemically defined medium with variations in the amounts of phosphates and in pH (4.00 to 9.00). All strains of a given species demonstrated similar acid phosphatase activities after growth in the presence or absence of phosphates. In contrast, alkaline phosphatase was produced only by some species, and it was also strongly influenced by the presence of either glycerophosphate or Pi in the medium. There were four patterns of phosphatase activity, depending on the properties of the alkaline phosphatase. Phosphatase activity can be a valuable characteristic for inclusion in tests for the identification of enterobacteria.

Novel toluidine derivatives are described. Some of them showed an interesting in vitro activity against Mycobacterium tuberculosis, M. smegmatis, M. marinum, M. gordonae, and M. avium. Some of them were more active than Streptomycin and Isoniazid, which were used as controls, against M. avium and M. gordonae. In particular, we confirm the good activity of biphenyl derivatives.