Broth Culture Filtrates Research Articles

Pleurotus ostreatus is a potential biological control agent of root-knot nematodes in eggplant (Solanum melongena)

IntroductionThe management of root-knot nematodes has predominantly been based on use of chemicals, which are detrimental to the environment and human health. Biological control provides alternative management. This study evaluated the potential of using Pleurotus ostreatus, an edible mushroom species to control Meloidogyne spp. in eggplants.MethodsIn vitro, the mortality of juveniles (J2) of Meloidogyne spp. were assessed i) P. ostreatus - water suspension with actively growing mycelia, and ii) different dilutions of P. ostreatus PDB broth culture filtrates. In the screen house nematicidal potential of P. ostreatus was tested on eggplants using artificially inoculated soils in a screen house. To attain this, juveniles (J2) of Meloidogyne spp. were inoculated at the base of plants in pots containing P. ostreatus colonized millet grains mixed in 3 kg of soil. The galling index (GI) (scale of 0 to 5), root growth and nematode populations in the different treatments were assessed.Results and discussionMortality of nematodes in the P. ostreatus - water suspension significantly increased with time, reaching over 88% at 48 h and 95% at 72 h. Mortality in undiluted filtrate was consistently significantly higher than the diluted filtrates and control without P. ostreatus. Mortality in the undiluted filtrate increased to 95% at 48 h. When 50 g of P. ostreatus-millet culture was mixed with 3 kg autoclaved pot soil, a GI of 0.95 was observed, dropping to 0.70 when the inoculum was doubled to 100 g. For the treatment without P. ostreatus, a high GI of 2.4 was scored. A significant difference in eggplant root growth and nematode population at (p = 0.02) was found across the treatments. The findings from this study for both in-vitro and pot assay suggest that P. ostreatus and its substrate are potential biological control agents for plant parasitic nematodes in eggplants.

Chaetoglobosin A contributes to the antagonistic action of Chaetomium globosum strain 61239 toward the apple Valsa canker pathogen Cytospora mali.

Apple Valsa canker (AVC) weakens apple trees and significantly reduces apple production in China and other East Asian countries. Thus far, very few AVC-targeting biocontrol resources have been described. Here, we present a thorough description of a fungal isolate (Chaetomium globosum, 61239) that has strong antagonistic action toward the AVC causal agent Cytospora mali. Potato dextrose broth (PDB) culture filtrate of strain 61239 completely suppresses the mycelial growth of C. mali on PDA, and strongly constrained the development of AVC lesions in in vitro infection assays. UPLC and HPLC-MS/MS investigations supported that strain 61239 produces Chaetoglobosin A, an antimicrobial metabolite that inhibits C. mali. Using genome sequencing, we discovered a gene cluster in strain 61239 that may be responsible for chaetoglobosin A production. Two of the cluster's genes - cheA, a PKS-NRPS hybrid enzyme, and cheB, an enoyl reductase - were individually silenced, which significantly decreased chaetoglobosin A accumulation as well as the strain's antagonistic activity against C. mali. Together, the findings of our investigation illustrate the potential use of Ch. globosum for the management of AVC disease and emphasize the significant contribution of chaetoglobosin A to the antagonistic action of strain 61239.

Optimization of the antifungal metabolite production in Streptomyces libani isolated from northern forests soils in Iran.

Background and Purpose: Soil bacteria have extreme population diversity among natural sources and are able to produce a wide array of antifungal metabolites. This study aimed to isolate and identify the bioactive metabolite-producing bacteria from forest soils and evaluate their antimicrobial potent against some pathogenic organisms.Materials and Methods: In this study, soil samples were screened for antifungal activity against Aspergillus fumigatus on glucose-yeast extract (GY) agar using a visual agar plate assay method. All growing bacteria were examined for antifungal activity, and antagonistic bacteria were identified based on 16S ribosomal RNA sequence analysis. For optimization of the production of antifungal bioactive metabolites, inhibitory bacteria were cultured on different culture conditions, including media, pH, temperature, and incubation time. Results: In total, 110 bacterial strains were isolated from the forest soils and four species with high antifungal activity were identified as Streptomyces libani, Streptomyces angustmyceticus, Bacillus subtilis, and Sphingopyxis spp. on the basis of 16s ribosomal RNA sequencing. Dichloromethane extract of the starch casein broth culture filtrate of the S. libani (incubated at 30° C for five days) showed strong antifungal activity against A. fumigatus, Aspergillus niger, and Aspergillus flavus. Conclusion: Based on the results, forest soils contain organisms with antifungal activity and could be considered as a good source for novel antifungal metabolites as effective and safe therapeutics.

Hydrocarbon Degradation and Enzyme Activities of Aspergillus oryzae and Mucor irregularis Isolated from Nigerian Crude Oil-Polluted Sites.

Many free-living saprobic fungi are nature recruited organisms for the degradation of wastes, ranging from lignocellulose biomass to organic/inorganic chemicals, aided by their production of enzymes. In this study, fungal strains were isolated from contaminated crude-oil fields in Nigeria. The dominant fungi were selected from each site and identified as Aspergillus oryzae and Mucor irregularis based on morphological and molecular characterization, with site percentage incidences of 56.67% and 66.70%, respectively. Selected strains response/tolerance to complex hydrocarbon (used engine oil) was studied by growing them on Bushnell Haas (BH) mineral agar supplemented with the hydrocarbon at different concentrations, i.e., 5%, 10%, 15%, and 20%, with a control having dextrose. Hydrocarbon degradation potentials of these fungi were confirmed in BH broth culture filtrates pre-supplemented with 1% engine oil after 15 days of incubation using GC/MS. In addition, the presence of putative enzymes, laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) was confirmed in culture filtrates using appropriate substrates. The analyzed fungi grew in hydrocarbon supplemented medium with no other carbon source and exhibited 39.40% and 45.85% dose inhibition response (DIR) respectively at 20% hydrocarbon concentration. An enzyme activity test revealed that these two fungi produced more Lac than MnP and LiP. It was also observed through the GC/MS analyses that while A. oryzae acted on all hydrocarbon components in the used engine oil, M. irregularis only degraded the long-chain hydrocarbons and BTEX. This study confirms that A. oryzae and M. irregularis have the potential to be exploited in the bio-treatment and removal of hydrocarbons from polluted soils.

Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

BackgroundThe fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)—secreted, membrane-bound nanoparticles containing molecular cargo—being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes.ResultsZymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC–MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs.ConclusionWe have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

Natural variation of root lesion nematode antagonism in the biocontrol fungus Clonostachys rosea and identification of biocontrol factors through genome-wide association mapping.

Biological control is a promising approach to reduce plant diseases caused by nematodes to ensure high productivity in agricultural production. Large‐scale analyses of genetic variation in fungal species used for biocontrol can generate knowledge regarding interaction mechanisms that can improve efficacy of biocontrol applications. In this study, we performed a genome‐wide association study (GWAS) for in vitro antagonism against the root lesion nematode Pratylenchus penetrans in 53 previously genome re‐sequenced strains of the biocontrol fungus Clonostachys rosea. Nematode mortality in C. rosea potato dextrose broth (PDB) culture filtrates was highly variable and showed continuous variation (p < .001) between strains, indicating a polygenic inheritance. Twenty‐one strains produced culture filtrates with higher (p ≤ .05) nematode mortality compared with the PDB control treatment, while ten strains lowered (p ≤ .05) the mortality. The difference in in vitro antagonism against P. penetrans correlated with antagonism against the soybean cyst nematode Heterodera glycines, indicating lack of host specificity in C. rosea. An empirical Bayesian multiple hypothesis testing approach identified 279 single nucleotide polymorphism markers significantly (local false sign rate < 10–10) associated with the trait. Genes present in the genomic regions associated with nematicidal activity included several membrane transporters, a chitinase and genes encoding proteins predicted to biosynthesize secondary metabolites. Gene deletion strains of the predicted nonribosomal peptide synthetase genes nps4 and nps5 were generated and showed increased (p ≤ .001) fungal growth and conidiation rates compared to the wild type. Deletion strains also exhibited reduced (p < .001) nematicidal activity and reduced (p ≤ .05) biocontrol efficacy against nematode root disease and against fusarium foot rot on wheat. In summary, we show that the GWAS approach can be used to identify biocontrol factors in C. rosea, specifically the putative nonribosomal peptide synthetases NPS4 and NPS5.

Antagonistic activity of cowshed Bacillus sp. bacteria against aflatoxigenic and sclerotic Aspergillus flavus

Mycotoxins produced by many food spoilage fungi cause serious damage to human as well as to livestock. Aflatoxins are one such group of mycotoxins produced by Aspergillus flavus in many agricultural and food products including rice. The fungus can virtually grow in any environment and also produces resistive structures such as sclerotia to overcome unfavourable environmental conditions. Hence, the need of the time is to control this toxigenic and sclerotic fungus through an eco-friendly approach. In the current study four biocontrol bacteria belonging to Bacillus species were isolated from Indian cow shed environment and these bacteria could efficiently control not only the mycelia growth of A. flavus but also the germination and growth of sclerotia. Both active bacterial culture broth and cell free culture filtrate could limit the growth of the fungus up to more than 90%. Older culture broth and filtrate lost their inhibition efficiency. Unlike many similar studies the current investigation emphasizes the importance of cowshed environment instead of cow dung and the control of sclerotia instead of fungal spores.

Studying the Optimum Conditions of ‎Hygromycin B Production and Detect their ‎Toxicity

Hygromycin B was extracted with ethyl acetate, which separates organic phase from aqueous phase in the broth culture filtrate, only the aqueous phase showed significant antimicrobial activity by using agar well diffusion technique. At a concentration of 25mg/ml (as crude extract), this phase excreted its activity against the test microorganisms which include; one G(+) bacteria (Staphylococcus aureus), five G(–) bacteria (Pseudomonas aeruginosa , Proteus mirabilis, Escherichia coli , Klebsiella pneumoniae, Salmonella typhi) and one yeast (Saccharomyces cerevisiae). &#x0D; After detecting the aminoglycoside hygromycin B by the Thin Layer Chromatography (TLC) method to ensure presence of the antibiotic, same flow rate (Rf) value (0.357) as that of the standard hygromycin B was obtained.&#x0D; Results of the optimization conditions showed that the highest antimicrobial activity of hygromycin B was obtained at a medium pH of 8 and incubation temperature of 35°C for 10 days. When the toxicity of hygromycin B crude extract under such conditions was examined on mice liver, a mild effects were appeared

Вплив умов зберігання фільтратів бульйонних культур пробіотиків на їхню протимікробну активність

Nowadays there is no standard efficient technology for long-term storage of metabolite-containing products of microbial origin as well as no data about the advantages and expediency of using one or another method of storage for these products. Determining the optimal storage conditions will prevent the loss of biological activity of metabolites during storage both for scientific research and production. Metabolites were obtained by isolation from the producers by means of centrifugation and filtration after culturing probiotics in a liquid nutrient medium. We studied antimicrobial activity of filtrates of probiotic culture liquids with various inoculation doses right after obtaining and storage for 60 days in liquid, frozen and frozen-dried state at (4 ± 1)°C, (–23 ± 1)°C and under hypothermic conditions (4 ±1)°C, respectively. The storage of probiotic broth culture filtrates under the mentioned above conditions did not result in a significant reduction of their antimicrobial activity. The obtained results can be used when developing commerciol synbiotics. Probl Cryobiol Cryomed 2017; 27(4): 311 – 321

Screening of soil samples for isolation of lipase producing organisms

Lipases, falling under serine ester hydrolases can catalyse both the hydrolysis of lipid and long chain triglycerol synthesis. Lipases find wide usage in industry especially in food industry, detergents, leather industry, environmental management, pharmaceutical industry and as biosensor. Bacteria are the most common source of lipases. Soil samples from around petroleum waste, dairy farm and paddy field from in and around Manipal were collected. The soil samples were studied for physical characteristics. Tributyrin agar media was used for screening lipolytic bacteria. One gram of soil sample from various sources was dissolved in 50mL of sterile water separately. The stock solutions were used for log dilution from 10−1 to 10−4. Last two dilutions of each sample were mixed with tributyrin agar media and plated in petri dishes to facilitate growth of lipolytic bacteria. Morphological characteristics of grown colonies were recorded and they were isolated and preserved on tributyrin agar slants. Morphology of isolated organisms was studied. Lipolytic activity was tested in cell free filtrate of broth culture for each isolate using cupric acetate/pyridine reagent at 715 nm. Enzyme activity was measured using standard curve prepared with stearic acid. A total of 12 different colonies (4 from each sample) were isolated and studied. Among the different isolates, many were found to be spherical, non-motile and Gram positive. One isolate from dairy farm showed promising lipolytic activity. Further studies on identification of the organism and optimization parameters are to be carried out.

Effects of crude extracted proteins from supernatant of BCF-P and EGF on acid secretion

In order to compare the in vitro effects of crude extracts of broth culture filtrate protein (BCF-P) from the supernatant of an H. pylori liquid culture and epidermal growth factor (EGF) on acid secretion by isolated rabbit gastric parietal cells. Isolated rabbit gastric parietal cells were incubated with BCF-P or EGF for 1 and 12 h. Morphological changes were assessed by electron microscopy, and 14C-aminopyrine (14C-AP) uptake and H+-K+ATP mRNA expression were measured. At 50 µg/mL, BCF-P stimulated acid secretion by parietal cells (P<0.05) at 1 h but not at 12 h. At 100 µg/mL, BCF-P significantly inhibited histamine-stimulated acid secretion (P<0.05) and decreased H+- K+ATP mRNA expression in parietal cells, but it was reversed after EGF exposure. Crude extracted proteins from BCF-P damaged the inner cell membrane structure of parietal cells, reduced H+-K+ATP gene expression, and inhibited acid secretion, activities that differed markedly from those of EGF.

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori.

BackgroundHelicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection.ResultsIn the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD50 lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes.ConclusionsG. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients’ disease status.

Effects of broth culture filtrate protein of VacA+ Helicobacter pylori on the proliferation and apoptosis of gastric epithelial cells

Background Infection with Helicobacter pylori (H. pylori) may lead to chronic inflammation of the stomach epithelium, mucosal atrophy, imbalance of proliferation and apoptosis of epithelial cells; resulting in chronic gastritis, peptic ulcer, gastric cancer, and many other clinical outcomes. Why and how H. pylorus leads to gastric cancer is not clear yet. Through in vitro experiments, this study evaluated the effects of broth culture filtrate protein (BCF-P) from the supernatant of liquid culture media of H. pylori on proliferation and apoptosis of immortalized human gastric epithelial cell lines (GES-1) and gastric cancer cell lines (AGS). Methods For the study, GES-1 and AGS cell lines mix with BCF-P and epidermal growth factor (EGF). MTT assay and flow cytometry (FCM) determined the levels of proliferation and apoptosis. Detected expression levels of cyclooxygenase-2 (COX-2) and Fas mRNA by reverse transcription (RT)-PCR. Also did analysis of the effects of BCF-P on epidermal growth factor receptor (EGFR) tyrosine kinase activity of GES-1 and AGS cells by non-radioactive enzyme-linked assay. The Student’s t test and one-way analysis of variance (ANOVA) were used for statistical analysis. Results BCF-P inhibited proliferation of GES-1 and AGS cells in a concentration-dependent manner. The inhibition rates are respectively 68.7% in AGS and 61.4% in GES-1. With the same dose and time for inhibiting the proliferation, BCF-P failed to induce apoptosis of GES-1 and AGS cells. Effects of BCF-P reduced the expression of Fas mRNA of GES-1 and AGS cells (P &lt;0.05). This is consistent with the effects of EGF. BCF-P reduced the expression of COX-2 mRNA of AGS cells (P &lt;0.05). This is opposite to the effects of EGF (P &lt;0.05). Effects of BCF-P improved more than three times the EGFR tyrosine kinase activity of GES-1 and AGS cells. Conclusions BCF-P inhibited the proliferation of AGS and GES-1 cells in vitro, unrelated to apoptosis. Effects of BCF-P on gastric epithelial cells in vitro are not equivalent to that of EGF.

Culture and physiological variability in Rhizoctonia solani, responsible for foliar and lesions on aerial part of soybean

Foliar blight of soybean is one of the major fungal diseases. Rhizoctonia solani isolated from soybean growing in tarai regions of Uttarakhand. Six isolates of R. solani has been characterized on the basis of cultural and physiological nature such as colony diameter, growth, colour and sclerotia formation were recorded. Potato Dextrose Agar (PDA) was found best for growth and development. Two isolates (Lakhimpur and Pantnagar) covered the whole plates (90 mm) in 48 hrs. However, maximum number of sclerotia and weight was recorded on Czapek Dox agar medium. Overall radial growth supporting is recorded Corn Meal Agar Medium. Varied range of temperatures i.e. 10, 15, 25, 30, 35 and 400C was tested and found better growth of different isolates of R. solani at 10 - 400C, with an optimum growth temperature at 300C. Isolates were grown on five broth media (Asthana &amp; Hawkers, Potato Dextrose Agar, Czapek’s Dox Agar, Corn Meal Agar and Richards Agar) for fresh, dry weight and oat meal broth culture filtrates of all isolates was used in phytotoxic effects. It recorded that maximum fresh and dry weight was observed on corn meal agar medium. The maximum reduction in radical and plumule length of germinating seeds were recorded in Haldichaur isolate.

Helicobacter pylori: molecular detection of vacA gene and vacuolating activity in human gastric adenocarcinoma cells.

Helicobacter pylori strains secrete a vacuolating cytotoxin (VacA), plays an important role for the development of peptic ulcer disease and gastro-duodenal diseases. vacA gene is responsible to regulate the activity of the vacuolating cytotoxin. The objective of this study was molecular detection of vacA gene and observes the vacuolating activity on human gastric adenocarcinoma (AGS) cells. H. pylori vacA gene was determined by polymerase chain reaction. Vacuolation activity of VacA toxin in broth culture filtrates was assessed in AGS cells and quantified by neutral uptake assay. Different concentration dosages of VacA and incubation time were used in the measurement of the vacuolating activity on AGS cells. The results showed that VacA toxin could stimulate vacuolating activity on AGS cells with minimum concentration 1.0 μg/ml from both of s1m1 and s1m2 alleles (vacA gene). The toxin produced optimal reaction at 5.0 μg/ml with significant differences observed between the alleles. The results also showed that both alleles commenced the vacuolating activity at the minimum of 3 hr incubation time, and the activity showed in time-dependent manner. Optimal concentration of VacA toxin (s1m1 allele) causes more interaction with AGS cell producing more vacuolating activities. Time-dependent vaculation of both alleles might allow H. pylori for persistent infection without rapid destruction of gastric cells might promote gastro-duodenal diseases. The study provided us better understanding of the pathogenesis of the diseases associated with H. pylori infection which is an emerging problem in developing countries.

The effects of cagA gene positive Hp culture filtrates on the growth of GES-1

Objective To study the effects of CagA gene positive Hp culture filtrates on the growth of GES-1. Methods There were three groups： CagA gene positive Hp group； CagA negative group was cultivated then the Hp culture filtrates were prepared. The GES-1 cells were treated with the different culture filtrates for one month. GES-1 cells were then examined by microscopy to observe morphologic changes， growth caculations were performed to test cell growth by MTT， single cell microgel electrophoresis (SCGE) was used to detect cellular DNA damage. Blank broth filtrates was used for contrast group. Results GES-1 cells treated with CagA gene positive broth culture filtrates of Hp showed dyskaryosis， hypertrophy of nucleolus and karyokinesis. growth caculations showed GES-1 proliferated actively when treated by CagA gene positive broth culture filtrates. In this group， comet assay showed 41.2% comet cells in GES-1 cells while in CagA negative group， the number was 12.5%， significantly lesser than the CagA positive filtrates(P＜0.05). Conclusions CagA gene plays an important role in the changes of the morphology and growth character of tumor cells in human gastric epithelial treated by Hp filtrates. DNA damage may be one of the mechanisms of the changes of growth character.

Molecular Alterations in Fibroblasts Exposed to Helicobacter pylori Broth Culture Filtrate: A Potential Trigger of Autoimmunity?

Molecular Alterations in Fibroblasts Exposed to <i>Helicobacter pylori</i> Broth Culture Filtrate: A Potential Trigger of Autoimmunity?

Assay of antioxidant potential of two Aspergillus isolates by different methods under various physio-chemical conditions.

The objective of this work was to screen fungi isolated from soil of different areas of Punjab, India for antioxidant activity by dot blot assay and around 45% of fungal isolates demonstrated antioxidant potential. Two selected strains of Aspergillus spp (Aspergillus PR78 and Aspergillus PR66) showing quantitatively best antioxidant activity by DPPH assay were further tested for their reducing power, ferrous ion and nitric oxide ion scavenging activity, FRAP assay and total phenolic content. Different physio-chemical parameters were optimized for enhancement of the activity. This revealed stationary culture grown for 10 days at 25 oC at pH 7 to be the best for antioxidant activity. Sucrose in the medium as carbon source resulted in highest antioxidant activity. Sodium nitrate, yeast extract, and peptone were good sources of nitrogen but sodium nitrate was the best among these. The extraction of the broth culture filtrates with different solvents revealed ethyl acetate extract to possess the best antioxidant activity. The activity as expressed by ethyl acetate extract of Aspergillus PR78 was equally effective as that of commonly used antioxidant standard, ascorbic acid.

Evidence for Transepithelial Dendritic Cells in Human H. pylori Active Gastritis

Despite extensive experimental investigation stressing the importance of bacterial interaction with dendritic cells (DCs), evidence regarding direct interaction of Helicobacter pylori or its virulence products with DCs in the human gastric mucosa is lacking. Human gastric mucosa biopsies, with or without H. pylori infection and active inflammation, were investigated at light and electron microscopy level with immunocytochemical tests for bacterial products (VacA, urease, outer membrane proteins) and DC markers (DC-SIGN, CD11c, CD83) or with the DC-labeling ZnI(2)-OsO(4 )technique. Parallel tests with cultured DCs were carried out. Cells reproducing ultrastructural and cytochemical patterns of DCs were detected in the lamina propria and epithelium of heavily infected and inflamed (but not of normal) mucosa, where DC luminal endings directly contact H. pylori and take up their virulence products. Cytotoxic changes (mitochondrial swelling, cytoplasmic vacuolation, autophagy) were observed in intraepithelial DCs and reproduced in cultured DCs incubated with H. pylori broth culture filtrates to obtain intracellular accumulation of VacA and urease. Granulocytes were also seen to contact and heavily phagocytose luminal H. pylori, while macrophages remained confined to basal epithelium, though taking up bacteria and bacterial products. Human DCs can enter H. pylori-infected gastric epithelium, in association with other innate immunity cells, to take up bacteria and their virulence products. This process is likely to be important for bacterial sensing and pertinent immune response; however, it may also generate DC cytotoxic changes potentially hampering their function.

Effects of different Helicobacter pylori culture filtrates on growth of gastric epithelial cells

To study the effects of different Helicobacter pylori (H pylori) culture filtrates on growth of gastric epithelial cells. Broth culture filtrates of H pylori were prepared. Gastric epithelial cells were treated with the filtrates, and cell growth was determined by growth curve and flow cytometry. DNA damage of gastric epithelial cells was measured by single-cell microgel electrophoresis. Gastric epithelial cells proliferated actively when treated by CagA-gene-positive broth culture filtrates, and colony formation reached 40%. The number of cells in S phase increased compared to controls. Comet assay showed 41.2% comet cells in GES-1 cells treated with CagA-positive filtrates (P < 0.05). CagA-positive filtrates enhance the changes in morphology and growth characteristics of human gastric epithelial tumor cells. DNA damage maybe one of the mechanisms involved in the growth changes.