Effects of temperature and medium composition on inhibitory activities of gossypol‐related compounds against aflatoxigenic fungi

Aims

To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.

Methods and Results

The compounds were tested at a concentration of 100 μg ml^?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.

Conclusions

Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.

Significance and Impact of the Study

Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.     

Development of a chemically defined medium for the production of enterolysin A from Enterococcus faecalis B9510

Aim

This study aimed to develop a simplified chemically defined medium that could sustain the growth and bacteriocin (enterolysin A) production by Enterococcus faecalis B9510.

Methods and Results

The nutritional requirements of E. faecalis B9510 in a chemically defined medium were determined by single omission experiments. It was observed that eight amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, tryptophan and valine), three B vitamins (nicotinic acid, Ca‐pantothenic acid and pyridoxal) and magnesium sulphate were essential for growth. Based on this information, a Simplified Defined Medium (SDM) was formed consisting of 26 components. Comparison of SDM with M‐17 showed that growth and bacteriocin production in SDM was similar to that in M‐17. The bacteriocin from SDM was then purified by ultrafiltration. The retentate of ultrafiltration step was analysed by SDS‐PAGE and the results showed a single active band in the gel, which was excised and analysed by mass spectrometry, which indicated that the active band was enterolysin A, a cell wall degrading bacteriocin.

Conclusions

A simplified defined medium can be formulated for the growth and bacteriocin production by Enterococcus faecalis, whose efficiency is comparable with that of a complex commercial medium.

Significance and Impact of the Study

The development of such a medium can be useful for bacteriocin production and subsequent purification in a simplified manner and, therefore, helpful in the identification of novel bacteriocins.     

Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei

Background  

Methods for the extraction of DNA from filamentous fungi are frequently laborious and time consuming because most of the available protocols include maceration in liquid nitrogen after the mycelium has been grown in a liquid culture. This paper describes a new method to replace those steps, which involves the growth of the mycelium on cellophane disks overlaid on solid medium and the use of glass beads for cell wall disruption.     

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat‐cured lime oil

Aims

This study aimed to examine heat curing effect (30–100°C) on antifungal activities of lime oil and its components (limonene, p‐cymene, β‐pinene and α‐pinene) at concentrations ranging from 100 to 300 μl ml^?1 against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata).

Methods and Results

Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 μl ml^?1 with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 μl ml^?1 lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions.

Conclusions

Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge.

Significance and Impact of the Study

Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.     

Development of a serum-free medium for <Emphasis Type="Italic">in vitro</Emphasis> expansion of human cytotoxic T lymphocytes using a statistical design

Background  

Serum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis.     

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

Background  

During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved.     

Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium

Background  

Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium.     

The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> becomes evident only in growing bacteria

Background  

We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida.     

α1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro

Background  

Tumor microenvironment, which is largely affected by inflammatory cells, is a crucial participant in the neoplastic process through promotion of cell proliferation, survival and migration. We measured the effects of polymorphonuclear neutrophil (PMN) conditioned medium alone, and supplemented with serine proteinase inhibitor α-1 antitrypsin (AAT) or its C-terminal fragment (C-36 peptide), on cultured lung cancer cells.     

Characterization of proton production and consumption associated with microbial metabolism

Background  

Production or consumption of protons in growth medium during microbial metabolism plays an important role in determining the pH of the environment. Such pH changes resulting from microbial metabolism may influence the geochemical speciation of many elements in subsurface environments. Protons produced or consumed during microbial growth were measured by determining the amount of acid or base added in a 5 L batch bioreactor equipped with pH control for different species including Escherichia coli, Geobacter sulfurreducens, and Geobacter metallireducens.     

<Emphasis Type="Italic">PGM2</Emphasis> overexpression improves anaerobic galactose fermentation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium.     

Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment

Background  

Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function.     

The role of bone marrow mononuclear cell-conditioned medium in the proliferation and migration of human dermal fibroblasts

Background

Several recent studies have demonstrated the great potential of bone marrow cells in regenerative medicine, not only for their ability to differentiate to match a damaged cell type, but also because they synthesize and release various growth factors and cytokines.We examined the effect of bone marrow cell-conditioned medium in the healing process, especially in terms of fibroblast proliferation and migration.

Methods

These in vitro studies consisted of co-culture (without direct contact) of dermal fibroblasts with mononuclear bone marrow cells and the use of conditioned medium obtained from these cultures in a scratch wound model.

Results

Mononuclear cells were found to increase the proliferation of fibroblasts, and the conditioned medium showed a stimulatory effect on the migration of fibroblasts.

Conclusion

When considered together with the observed increase in growth factor levels in conditioned medium, it appears that these cells act through a paracrine mechanism.

     

HDACs and the senescent phenotype of WI-38 cells

Background  

Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells.     

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium

Background

Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.

Principal Finding

The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×10⁶ cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×10⁸ pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.

Conclusions

The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.     

Chemically defined medium and <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

C. elegans has been established as a powerful genetic system. Use of a chemically defined medium (C. elegans Maintenance Medium (CeMM)) now allows standardization and systematic manipulation of the nutrients that animals receive. Liquid cultivation allows automated culturing and experimentation and should be of use in large-scale growth and screening of animals.     

Production of recombinant antibody fragments in <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Background  

Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.     

Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris

Background  

Secretion of heterologous proteins depends both on biomass concentration and on the specific product secretion rate, which in turn is not constant at varying specific growth rates. As fed batch processes usually do not maintain a steady state throughout the feed phase, it is not trivial to model and optimize such a process by mathematical means.     

Cytochrome P450 aromatase expression in human seminoma

Background  

The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis.