The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil

Our objective was to determine if 4-methylumbelliferyl-labelled enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF) N-acetyl-β-d-glucosaminide and MUF β-d-lactoside were used for the detection and quantification of β-N-acetylglucosaminidase (EC 3.2.1.30) (NAGase) and endo 1,4-β-glucanase (EC 3.2.1.4)/cellobiohydrolase (EC 3.2.1.91) (CELase), respectively. Culture screenings on solid media showed a widespread ability to produce NAGase among a taxonomically diverse selection of fungi on media with and without added chitin. NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of two fungus-specific indicator molecules, 18:2ω6 phospholipid fatty acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil, but no correlation was found with ergosterol-based estimates of fungal biomass.The determination of enzyme activities is a simple approach to the study of microbially mediated processes within the soil environment. Thus, soil enzyme activities have been interpreted as indirect measures of microbial biomass, rhizosphere effects, soil productivity, and mineralization potential of naturally occurring substrates or xenobiotics (4). However, few studies have attempted to correlate soil enzyme activities with the presence and activities of specific components of the microbial community. The ability of soil-inhabiting fungi to produce a range of enzymes capable of degrading complex litter substances could make the use of an enzymatic approach to study soil fungal populations possible. These enzymes must be specific for fungal presence and activity. In one study of chitinase in soil (24), chitinase activity and the number of fungal propagules in chitin-amended soils were strongly correlated. The same correlation was not found for actinomycetes or bacteria. Thus, chitinase activity appears to be a suitable indicator of actively growing fungi in the soil. The hydrolysis of cellulose requires the interaction of a number of hydrolases produced by cellulolytic microorganisms. A major role is played by the cellulase system, which consists of several distinct enzymes that are produced by a large number of microorganisms, including fungi, actinomycetes, and bacteria. However, fungi have been suggested as the predominant source of β-d-glucosidase (EC 3.2.1.21) (16, 17) and endo 1,4-β-glucanase (EC 3.2.1.4) (23) activity in soils.Fluorogenic 4-methylumbelliferyl (MUF)-labelled enzyme substrates have been introduced for process-oriented studies in aquatic systems (3, 18) and, more recently, in peatlands (11). MUF substrates have been used to assay cell-bound activities in pure cultures of fungi, as the soluble substrate can enter the cell wall, making periplasmic enzyme activity detectable (15). These substrates have been used to detect fungal chitinolytic activities (17a) and cellulases (6) in vitro. The substrates may be added to environmental samples and, when hydrolyzed, release 4-methyl-umbelliferone (4-MU), which fluoresces and can be quantified in nanomolar concentrations (3).A variety of methods to quantify fungi in soil have been described. The techniques include direct microscopic observation and extraction of fungus-specific indicator molecules such as glucosamine or ergosterol (9). More recently, the phospholipid fatty acid (PLFA) 18:2ω6 has been proposed as an indicator of fungal biomass (7, 12). Our objectives in the present study were to determine if (i) components of chitinase and cellulase activities could be used as indicators of the presence and activity of fungal biomass and (ii) enzyme activities detected with specific MUF substrates in soil samples were correlated with the content of the fungus-specific indicator molecules 18:2ω6 PLFA and ergosterol.     

Relationships of the Insect-Pathogenic Order Entomophthorales (Zygomycota, Fungi) Based on Phylogenetic Analyses of Nuclear Small Subunit Ribosomal DNA Sequences (SSU rDNA)

We sequenced the nuclear small subunit of ribosomal DNA (SSU rDNA) from seven species within the insect-pathogenic order Entomophthorales. These sequences were aligned with other published SSU rDNA sequences and phylogenies were inferred using phenetic and cladistic methods. Based on three different phylogenetic methods the Entomophthorales (excludingBasidiobolus ranarum) is monophyletic;B. ranarumwas more closely related to chytrids from Chytridiales and Neocallimasticales than to Entomophthorales, as was proposed by Nagahamaet al.(Mycologia87:203–209, 1995). Nuclear characters (large nuclei containing conspicuous condensed chromatin and lack of a prominent nucleolus) were of predictive value for the monophyly of the family Entomophthoraceae. Conidial characters separate the Entomophthoraceae, which only includes obligate pathogens, into at least two lineages: one lineage with uninucleate conidia and another with multinucleate conidia. The two species ofConidiobolusstudied were paraphyletic in our analyses and only distantly related to each other. This information may prove to be important in the use of these fungi as biocontrol agents.     

Separation of metalloprotein complexes in serum by size-exclusion chromatography: Optimisation of the separation parameters retention behaviour and recovery employing radiotracers

A suitable procedure was developed for speciation analysis of metalloprotein complexes in serum using directly coupled size-exclusion chromatography and an element-specific detector. Two column matrices used for size-exclusion chromatography (TSK G 3000 SW and Asahipak GS 520) were investigated with respect to the recovery and retention behaviour for metalloprotein complexes. Optimisation of the separation parameters (buffer type, concentration, pH) was achieved by means of metalloprotein complexes marked with radiotracers. For speciation of serum the matrix in the Asahipak GS column is more efficient. Given optimal eluent characteristics (100 mM Tris, pH 7.4) the recovery of the elements investigated (sodium, calcium, iron and zinc) was 100%. Further, the retention behaviour (retention time, ratios of the peak areas) remained unchanged for several successive separations.     

Effect of some plant growth regulator treatments on apple fruit ripening

The activity of IAA oxidase (IAAox), peroxidases (POD), and polyphenoloxidases (PPO), as affected by different pre-harvest growth regulator treatments (ABA, AVG, NAA, PDJ), was determined in on-tree ripening apples (cv. Golden Delicious) before and during the ethylene climacteric. The production of ethylene was inhibited by AVG and delayed by NAA, whereas ABA and PDJ treatments caused, in the on-tree remaining fruits, a marked fruit drop and a decrease or a slight increase in ethylene levels respectively. While all treatments reduced POD activity, jasmonate increased IAAox and PPO activity. The inhibitory effect of NAA on all enzyme activity seems related to interference with C₂H₂ action or to a reduced sensitivity of the fruit abscission zone tissues to the hormone. The observed high fruit drop induced by ABA treatment made it impossible to detect differences in enzyme activity. AVG-treated fruits showed no substantial effects on IAAox and PPO activity in comparison to the control, a finding that seems to be related to a delay in all senescence processes caused by the very low level of the inhibited ethylene production. In control fruits IAAox activity increased during the initial ripening stages and decreased thereafter, POD activity increased throughout ripening and PPO showed little variation.     

Photosynthetic electrogenic events in native membranes of Chloroflexus aurantiacus. Flash-induced charge displacements in the acceptor quinone complex of the photosynthetic reaction center

Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. Tertiary structure modeling suggests that the redox-sensitivity of the chloroplast malate dehydrogenase (EC 1.1.1.82) is due to disulfide crosslinking of the carbon substrate and nucleotide-binding domains. Consistent with this suggestion, introduction of Cys residues in opposition to one another on the two domains of the Escherichia coli enzyme results in redox-sensitivity [Muslin EH et al. (1995) Biophys J 68: 2218-2223]. We have now substituted Cys residues into the bacterial malate dehydrogenase (EC 1.1.1.37) in positions that correspond more exactly to those postulated to be responsible for the redox-sensitivity of the chloroplast enzyme. The introduction of one pair of Cys residues renders the enzyme redox-sensitive, but the introduction of the alternate pair does not. Energy minimization calculations suggest that the difference in redox-sensitivity is consistent with differences in the energy required for formation of the disulfide bond.     

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in Saccharomyces cerevisiae

Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three‐ to tenfold improvements over wild‐type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single‐chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16‐fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180‐fold improvement over previous reports in the secretion of full‐length, functional, glycosylated human IgG₁. The production of full‐length IgG₁ at milligram per liter titers in a simple, laboratory‐scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. Biotechnol. Bioeng. 2009;103: 1192–1201. © 2009 Wiley Periodicals, Inc.     

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.