Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Grants from the National Technology Agency of Finland (No. 40168/03) and the Academy of Finland (No. 52104); travel grants from NorFA and the European Commission to the Laboratory of Dr. Ulf Thrane     

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases

Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis.     

Gas chromatographic-mass spectrometric method for the determination of alkylresorcinols in human plasma

Alkylresorcinols can be found in high amounts in whole grain cereals, especially in rye. Previously it has not been possible to measure alkylresorcinols in plasma. In this paper a validated gas chromatographic-mass spectrometric method for the quantitative determination of alkylresorcinols with chain lengths of C15:0, C17:0, C19:0, C21:0, and C23:0 in human plasma samples is presented. Other alkylresorcinols may be measured with the method as well, but their assay was not validated in this work. In this work also the amount of alkylresorcinol C25:0 was measured. The pretreatment of plasma samples consists of a simple incubation, an extraction with diethyl ether and a chromatographic purification before the GC-MS analysis. As internal standard an alkylresorcinol C20:0 was used. The validation of the method showed that it fulfilled the reliability criteria. Calibration graphs were linear over the range of 4.1-660pg per injection. The mean recovery percentage was 112+/-10.8%. Our results show that the alkylresorcinols are found in plasma in the same ratio, as found in rye grains, according to literature. The alkylresorcinols were in the unconjugated form. The total amounts of alkylresorcinols in two plasma samples analyzed here were 333 and 381nmol/L.     

Functional and anionic cellulose-interacting polymers by selective chemo-enzymatic carboxylation of galactose-containing polysaccharides

Carboxylated, anionic polysaccharides were selectively prepared using a combination of enzymatic and chemical reactions. The galactose-containing polysaccharides studied were spruce galactoglucomannan, guar galactomannan, and tamarind galactoxyloglucan. The galactosyl units of the polysaccharides were first oxidized with galactose oxidase (EC 1.1.3.9) and then selectively carboxylated, resulting in the galacturonic acid derivatives with good conversion and yield. The degrees of oxidation (DO) of the products were determined by gas chromatography-mass spectrometry (GC-MS). A novel feasible electrospray ionization-mass spectrometry (ESI-MS) method was also developed for the determination of DO. The solution properties and charge densities of the products were investigated. The interaction of the products with cellulose was studied by two methods, bulk sorption onto bleached birch kraft pulp and adsorption onto nanocellulose ultrathin films by quartz crystal microbalance with dissipation (QCM-D). To study the effect of the location of the carboxylic acid groups on the physicochemical properties, polysaccharides were also oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated reaction producing polyuronic acids. The chemo-enzymatically oxidized galacturonic polysaccharides with an unmodified backbone had a better ability to interact with cellulose than the TEMPO-oxidized products. The selectively carboxylated polysaccharides can be further exploited, as such, or in the targeted functionalization of cellulose surfaces.