Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

Marine Algae of the Genus Gracilaria as Multi Products Source for Different Biotechnological and Medical Applications.

BACKGROUND: Gracilaria has been shown to be an important source of marine bioactive natural biomaterials and compounds. Although there are no enough patents used Gracilaria worldwide, the current study tries to put the Gracilaria on the spot for further important patents in the future. OBJECTIVE: The current study investigates the pharmaceuticals and biochemical activity of Gracilaria because no previous studies have been carried out to examine the biochemical and pharmaceutical activates of Gracilaria from the Suez Canal of Egypt as an excellent source for bioactive compounds. METHODS: Different advanced experimental models and analytical techniques, such as cytotoxicity, total antioxidant capacity, anticancer, and anti-inflammatory profiling were applied. The phytochemical analysis of different constituents was also carried out. RESULTS: The mineral analysis revealed the presence of copper (188.3 ppm) and iron (10.07 ppm) in addition to a remarkable wealth of selenium and sulfur contents giving up to 36% of its dry mass. The elemental analysis showed high contents of sulfur and nitrogen compounds. The GCMS profiling showed varieties of different bioactive compounds, such as fatty acids, different types of carotenoids in addition to pigments, alkaloids, steroids. Many other compounds, such as carbohydrates and amino acids having antioxidant, anti-inflammatory, and antiviral activities, etc. were identified. The cytotoxicity activity of Gracilaria marine extract was very effective against cancerous cell lines and showed high ability as a potent antitumor due to their bioactive constituents. Specialized screening assays using two anticancer experimental models, i.e., PTK and SKH1 revealed 77.88% and 84.50% inhibition anticancer activity; respectively. The anti-inflammatory activities investigated using four different experimental models, i.e., COX1, COX2, IL6, and TNF resulted in 68%, 81.76%, 56.02% and 78.43% inhibition; respectively. Moreover, Gracilaria extracts showed potent anti-Alzheimer with all concentrations. CONCLUSION: Gracilaria proved to be a multi-product source of marine natural products for different biotechnological applications. Our recommendation is to investigate the Gracilaria bioactive secondary metabolites in order to create and innovate in more patents from current important seaweeds (Gracilaria).

Use of metal/metal oxide spherical cluster and hydroxyl metal coordination complex for descriptor calculation in development of nanoparticle cytotoxicity classification model.

Computational approaches have been suggested as an informative tool for risk assessment of nanomaterials. Nano (quantitative) structure-activity relationship, nano-(Q)SAR, models have been developed to predict toxicity of metal oxide (MOx) nanoparticles (NPs); however, the packing structure and cluster of nanoparticle have been included for the descriptor calculation in only two studies. This study proposed spherical cluster and hydroxyl metal coordination complex to calculate descriptors for development of nanoparticle cytotoxicity classification model. The model cluster was generated from metal (M) or MOx crystal structure to calculate physicochemical properties of M/MOx NPs and the hydroxyl metal coordination complex was used to calculate the properties of the metal cation in an aqueous environment. Data were collected for 2 M and 19 MOx NPs in human bronchial epithelial cell lines and murine myeloid cell lines at 100 µg/ml after 24 hours exposure. The model was developed with scaled HOMO energy of the model cluster and polarizability of the hydroxyl metal coordination complex, as reactivity of the particles and the cations explained cause of cytotoxic action by M/MOx NPs. As the developed model achieved 90.31% accuracy, the classification model in this work can be used for virtual screening of toxic action of M/MOx NPs.

Distribution of MACPF/CDC proteins.

Membrane Attack Complex/Perforin (MACPF) and Cholesterol-Dependent Cytolysins (CDC) form the MACPF/CDC superfamily of important effector proteins widespread in nature. MACPFs and CDCs were discovered separately with no sequence similarity at that stage being apparent between the two protein families such that they were not, until recently, considered evolutionary related. The breakthrough showing they are came with recent structural work that also shed light on the molecular mechanism of action of various MACPF proteins. Similarity in structural properties and conserved functional features indicate that both protein families have the same evolutionary origin. We will describe the distribution of MACPF/CDC proteins in nature and discuss briefly their similarity and functional role in different biological processes.

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes.

BACKGROUND: Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing. RESULTS: Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection. CONCLUSIONS: Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

Evaluation of cytotoxicity and mutagenicity of the benzodiazepine flunitrazepam in vitro and in vivo

Flunitrazepam (FNZ) is a sedative benzodiazepine prescribed for the short-term treatment of insomnia. However, there are concerns regarding possible carcinogenic or genotoxic effects of this medicine. Thus, the aim of this study was to evaluate the cytotoxic, clastogenic and aneugenic effects of FNZ in hepatoma cells from Rattus norvegicus (HTC) in vitro and in bone marrow cells of Wistar rats in vivo. These effects were examined in vitro following treatment with 0.2, 1.0, 5.0 or 10 μg/mL FNZ using a micronucleus test with a cytokinesis block or in vivo using a chromosomal aberration test following treatment with 7, 15 or 30 μg/mL/kg body weight. The results showed that the benzodiazepine concentrations tested were not cytotoxic, aneugenic or clastogenic. However, considering the adverse effects of using this benzodiazepine, more studies are required.

Flunitrazepam (FNZ) é um sedativo benzodiazepínico prescrito para o tratamento da insônia em curto prazo. Entretanto, existe a preocupação com relação aos possíveis efeitos carcinogênicos ou genotóxicos causados por este fármaco. Então, o objetivo deste estudo foi avaliar os efeitos citotóxicos, clastogênicos e aneugênicos do FNZ em células de hepatoma de Rattus norvegicus (HTC) in vitro e em células de medula óssea de ratos Wistar in vivo. Foram testadas as concentrações de 0,2, 1,0 e 10 μg/mL de FNZ pelo teste do micronúcleo com bloqueio de citocinese in vitro e 7, 15 e 30 μg/mL/kg de peso corpóreo para o teste de aberração cromossômica in vivo. Os resultados mostraram que as concentrações do benzodiazepínico testadas não foram citotóxicas, aneugênicas ou clastogênicas. Entretanto, considerando os efeitos adversos do uso deste benzodiazepínico, mais estudos são necessários.

Mutagenicity and DNA damage of bisphenol A and its structural analogues in HepG2 cells.

Environmental oestrogen bisphenol A (BPA) and its analogues are widespread in our living environment. Because their production and use are increasing, exposure of humans to bisphenols is becoming a significant issue. We evaluated the mutagenic and genotoxic potential of eight BPA structural analogues (BPF, BPAF, BPZ, BPS, DMBPA, DMBPS, BP-1, and BP-2) using the Ames and comet assay, respectively. None of the tested bisphenols showed a mutagenic effect in Salmonella typhimurium strains TA98 and TA100 in either the presence or absence of external S9-mediated metabolic activation (Aroclor 1254-induced male rat liver). Potential genotoxicity of bisphenols was determined in the human hepatoma cell line (HepG2) at non-cytotoxic concentrations (0.1 µmol L(-1) to 10 µmol L(-1)) after 4-hour and 24-hour exposure. In the comet assay, BPA and its analogue BPS induced significant DNA damage only after the 24-hour exposure, while analogues DMBPS, BP-1, and BP-2 induced a transient increase in DNA strand breaks.

Functional and phylogenetic characterization of Vaginolysin, the human-specific cytolysin from Gardnerella vaginalis.

Pore-forming toxins are essential to the virulence of a wide variety of pathogenic bacteria. Gardnerella vaginalis is a bacterial species associated with bacterial vaginosis (BV) and its significant adverse sequelae, including preterm birth and acquisition of human immunodeficiency virus. G. vaginalis makes a protein toxin that generates host immune responses and has been hypothesized to be involved in the pathogenesis of BV. We demonstrate that G. vaginalis produces a toxin (vaginolysin [VLY]) that is a member of the cholesterol-dependent cytolysin (CDC) family, most closely related to intermedilysin from Streptococcus intermedius. Consistent with this predicted relationship, VLY lyses target cells in a species-specific manner, dependent upon the complement regulatory molecule CD59. In addition to causing erythrocyte lysis, VLY activates the conserved epithelial p38 mitogen-activated protein kinase pathway and induces interleukin-8 production by human epithelial cells. Transfection of human CD59 into nonsusceptible cells renders them sensitive to VLY-mediated lysis. In addition, a single amino acid substitution in the VLY undecapeptide [VLY(P480W)] generates a toxoid that does not form pores, and introduction of the analogous proline residue into another CDC, pneumolysin, significantly decreases its cytolytic activity. Further investigation of the mechanism of action of VLY may improve understanding of the functions of the CDC family as well as diagnosis and therapy for BV.

[Rapid experimental rationale for a waste hazard class by cytotoxicity].

The paper presents the results of experimental and analytical studies substantiating a classification of waste hazard by the cytotoxicity indices. The authors have established a significant correlation between the substance toxicity values obtained in vivo and in vitro and show it possible to make an approximate forecast of the average lethal concentration of substances by the estimates made on cell cultures. The criteria for toxicological waste hazard, which are adequate to those for the hazards of chemicals by DL50, are given.

Multiple regression analysis as a tool for the identification of relations between semi-quantitative LC-MS data and cytotoxicity of extracts of the fungus Fusarium avenaceum (syn. F. arthrosporioides).

The cytotoxicity of methanolic extracts from rice cultures of 53 Fusarium avenaceum strains, which had been isolated from different host organisms in Northern Europe, Canada and Australia/New Zealand, was investigated in a rat hepatoma (H4IIE-W), porcine epithelial kidney (PK-15), foetal feline lung fibroblast, dog lymphoblast (D3447), and a human hepatocarcinoma (Hep G2) cell line using the Alamar Bluetrade mark assay. All extracts were screened for known fungal metabolites using high-performance liquid chromatography with photodiode array and mass spectrometric detection, and both known and unknown metabolites were semi-quantified. Known metabolites that were determined in the cultures include acuminatopyrone, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), antibiotic Y, aurofusarin, chlamydosporol, chlamydospordiol, enniatins, fusarin A and C, and moniliformin. Multiple regression analysis was used in order to relate fungal metabolites to the cytotoxicity of the extracts. Separate linear regression models were constructed for each cell line. Eleven different fungal metabolites were related to the cytotoxicity (P<0.05). Out of these, nine metabolites were siginificantly related to the cytotoxicity in only one of the five models, while two, namely enniatins and 2-AOD-3-ol, were significant contributors in three or four regression models, respectively. This paper describes how multiple regression analysis may be applied for the assignment of bioactivity/toxicity to the constituents of a multi-component mixture.

Detection of Pseudomonas aeruginosa type III antibodies in children with tracheostomies.

Pseudomonas aeruginosa is often cultured from the airways of children with tracheostomies. P. aeruginosa produces exotoxin A (ETA) and type III cytotoxins. This study tested the hypothesis that children with tracheostomies are colonized by P. aeruginosa that express these virulence factors and will have antibodies directed against these virulence factors, indicating infection rather than only colonization. A convenience sample of 30 patients, ranging in age from 2 months-22 years, was recruited. Serum was tested for the presence of antibodies to ETA and components of the type III system by Western blot analysis. Twenty-one of 39 patients (70%) had antibodies to components of the type III system. Fifteen of 30 (50%) were seropositive for ETA. Sera from patients who were antibody-positive for ETA were also seropositive for either ExoS or ExoU. Nine of 30 patients (30%) did not possess antibodies to ETA or components of the type III system. In conclusion, these data identified a seropositive reaction to P. aeruginosa cytotoxins in some patients with tracheostomies, suggestive of infection by cytotoxic strains of P. aeruginosa. Future studies will determine the utility of measuring seroconversion to these cytotoxins as an early indication of infection in children with tracheostomies.

Cloning and sequencing of cnf1 from Escherichia coli incriminated in mink and bovine colibacillosis.

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.

Toxin production by Campylobacter spp.

Of all the virulence factors that were proposed for Campylobacter jejuni and related species to cause disease in humans, the discovery of toxin production was the most promising but led to a rather confusing and even disappointing stream of data. The discussion of whether proteinaceous exotoxins are relevant in disease remains open. One important reason for this lack of consensus is the anecdotal nature of the literature reports. To provide a basis for an unbiased opinion, this review compiles all described exotoxins, compares their reported properties, and provides a summary of animal model studies and clinical data. The toxins are divided into enterotoxins and cytotoxins and are sorted according to their biochemical properties. Since many Campylobacter toxins have been compared with toxins of other species, some key examples of the latter are also discussed. Future directions of toxin research that appear promising are defined.

Cytolytic toxins from gram-negative bacteria.

Many Gram-negative bacterial pathogens synthesize cytolytic toxins as virulence factors. Most of these toxins generate pores in eukaryotic cell membranes, but there are apparently several different mechanisms of pore formation. Cytolysins of Gram-negative bacteria are usually synthesized as precursor proteins which are converted to the active toxins by modification or proteolytic processing. Such a requirement for activation is not common for cytolysins produced by Gram-positive bacteria. The extracellular secretion of cytolytic toxins from Gram-negative bacteria depends on specific transport systems.

Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes.

The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for Actinobacillus pleuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9, 10 and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxI operon will have the designations apxIC, apxIA, apxIB, and apxID for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII, ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular mass between 103 and 105 kDa. The genes of the apxII operon will have the designations apxIIC for the activator gene and apxIIA for the structural toxin gene. In the apxII operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxIB and apxID of the apxI operon. The designation ApxIII is proposed for the nonhaemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin III (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat).(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.

Properties of Vero cytotoxin-producing Escherichia coli of human origin of O serogroups other than O157.

The 48 Vero cytotoxin-producing Escherichia coli (VTEC) examined for properties associated with virulence were of human origin and represented 17 O serogroups other than O157 and O26. Only Vero cytotoxin production was common to all the strains. About 60% produced enterohemolysin and hybridized with the CVD419 probe derived from plasmid sequences of E. coli O157. Thirteen strains gave localized adherence (LA) to HEp-2 cells. All of these hybridized with the E. coli attaching and effacing (eae) gene probe and were positive in the fluorescence actin staining test, properties characteristic of strains that efface intestinal microvilli. A further 5 strains were eae probe-positive but did not give LA. None of the VTEC hybridized with a probe specific for the enteropathogenic E. coli adherence factor. Seven strains adhered to HEp-2 cells in a diffuse or aggregative pattern but did not hybridize with probes for these phenotypes. Non-O157 E. coli strains are diverse in their properties, although some may share virulence mechanisms with other diarrheogenic E. coli.