Removal of humic substances from soil DNA using aluminium sulfate

Direct extraction of soil DNA has become essential for the study of soil microorganisms. Humic substances co-extracted during DNA retrieval is a big problem because it greatly inhibits the enzymes involved in manipulating DNA. Popular commercial kits available for soil DNA extraction are unable to overcome this problem. Here we report an effective protocol for the removal of humic substance from soil DNA. The protocol involves flocculation of the humic substance by excessive Al(3+), then removal of superfluous Al(3+) via pH adjustment and finally release of soil microbial DNA by SDS lysis. This technique is superior to that employed by the UltraClean Soil DNA Kit and can be applied to a wide variety of soils.     

Content of auxin-inhibitor-and gibberellin-like substances in humic acids

The content of auxin-, inhibitors-and gibberellin-like substances in freeze-dried humic acids was studied. The results indicate the existence of growth-promoting substances in humic acids, possibly corresponding to IAA or its precursors. Inhibitory activity is more marked during germination than during the growth process. The inhibitory effect caused by high concentration of humic acids could be due to an auxin-like action rather than an input per se of growth inhibitors. A significant amount of both free and conjugated gibberellin-like substances were observed.     

Intensification of phenol biodegradation by humic substances

Phenol biodegradation was carried out in a batch system by the bacterial strain Cupriavidus metallidurans in the presence of potassium humate that was prepared by alkaline extraction from oxyhumolite. The experiments were focused on the assessment of the humate effect on biodegradation activity of the tested bacterial strain. The achieved results demonstrated that the humate has a positive influence on the biodegradation of phenol and reduces the incubation time necessary for phenol removal. Higher biodegradation rate and more intensive growth were observed during the cultivation in presence of humate in comparison to the cultivation without its addition. Adsorption of the humate on bacterial biomass was observed as well. Subsequently, a phenol biodegradation testing in a continuous-flow system using a biofilm reactor was also carried out. Although the reactor was inoculated by C. metallidurans only, the microbial composition under an aerobic non-aseptic condition during this long-term cultivation changed. The phenol removal efficiency obtained in the biofilm reactor was higher than 92% when phenol concentration in a treated medium was 1200 mg l⁻¹.     

Radioimmunoassay for antibodies in varicella-zoster virus serology.

The method of RIA for antibodies was employed with success in VZ virus serology. The method is suitable for testing VZ antibodies in the course of varicella or herpes zoster disease as well as for determining anamnestic titres. Its advantages are stability of antigen, objective reading of results and applicability to testing large serum sets.     

Biostimulant activity of humic substances extracted from leonardites

Background and aims

Biostimulants are natural compounds that enhance plant growth and plant nutrient use efficiency. In this study, biostimulant effects of humic substances (HS) extracted from leonardites were analysed on the metabolism of maize plants grown in hydroponic conditions.

Methods

HS extracted from four leonardites were tested for their auxin-like and gibberellin-like activities. Then, 11 day old maize seedlings were treated for 48 h with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L^?1) of HS. After sampling, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analysed.

Results

HS from leonardites had similar spectroscopic pattern, with small differences. The HS from the South Dakota lignite (HS_USA) had more carboxylic groups, whereas the three from Turkish mines had more aromatic and aliphatic structures. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols, p-hydroxybenzoic acid, p-coumarilic acid, and chlorogenic acid.

Conclusion

This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances.

     

The effect of fire on soil humic substances

Summary High temperature effects on soil humic substances were investigated both in natural and laboratory conditions. Differential Thermal Analysis and pyrolysis show that only temperatures above 250°C produce changes in the humic acid fraction. The mean change is a loss of weight due to decarboxylation. Isotachophoretic studies also show that temperatures higher than 250°C produce alteration in the separation pattern of the different subfractions due to changes in the ‘core’ or/and in the amount of carboxyl and hydroxyl groups. Isotachophoregrams done on soil samples extracted from an area before and after fire show no differences. This result is taken as an indication that there was no direct effect of the temperature on the soil humic substances.     

C-13 nuclear magnetic resonance spectroscopy of humic substances

Summary Evidence is presented from a preliminary study showing that C-13 N.M.R. spectroscopy is a useful new tool for the elucidation of the structure of soil organic matter.     

Chemical characterization of humic substances extracted from organic-waste-amended soils

Humic substances were extracted from a soil treated, in a 4-year experiment, at different rates with a sludge from anaerobic treatment of combined civil and industrial wastes, and with agricultural manure. Elemental and chemical analyses, molecular weight (MW) distribution and infrared (IR) spectroscopy were performed on the purified humic acids (HA). Organic wastes significantly increased the HA content of the treated soils and improved CEC. The C/N, C/H and C/O ratios of HA extracted from the original wastes showed a higher degree of humification for sludge than for manure. This difference was also noticed for the C/N ratio of soil humic extracts, indicating a faster humification process for the sludges in soil. The content of oxygen-containing functional groups was lower than the ‘model’ HA reported in the literature, and even more so for HA from sludges, reflecting their anaerobic formation. MW distribution and E₄/E₆ ratios showed that the sludge material had a higher molecular complexity than manure, supporting the high degree of humification attributed to the former. HA extracted from sludge-treated soils revealed a molecular dimension increasing with the application doses of waste material. Infrared spectra showed that HA formed in soils after waste additions reflected the chemical composition of the original organic material, which was rich in aliphatic groups and peptides for sludge and in carbohydrates for manure. On the basis of this study, it is concluded that not only are organic waste additions able to build up the HA content in soils but the HA formed assume the chemical characteristics and the degree of humification of the original material.     

Detection of apoptosis in vivo using antibodies against caspase-induced neo-epitopes

Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is still a widely used method, it is likely biased by sensitivity problems and observed-based variations. The availability of caspase-mediated neo-epitope-specific antibodies offers new tools for the detection of apoptosis in situ. Here, we discuss the use of immunohistochemical detection of cleaved caspase 3 and lamin A for the assessment of apoptotic cells in paraffin-embedded liver tissue. Furthermore, we evaluate the effect of tissue pretreatment and antigen retrieval on the sensitivity of apoptosis detection, background staining and maintenance of tissue morphology.     

Synthesis and physico-chemical properties of peptides in soil humic substances.

Soil humic substances (HS) are heterologous, polydispersive, and multi-functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with 13C-glucose or 15N-nitrate for 8.5 months. Evidence for random-coil peptidic structures in the labeled HS was obtained from 2-D nuclear magnetic resonance (NMR), pyrolysis gas chromatography-mass spectrometry (pyro-GC-MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid-state NMR data. Detailed 13C and 15N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC-MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre-existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.     

Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs

The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1-/- ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity.     

Functional studies using antibodies against orphanin FQ/nociceptin

Orphanin FQ/nociceptin (OFQ) is a recently discovered endogenous ligand for the novel opioid receptor-like receptor (ORL-1). There are numerous reports in the literature demonstrating paradoxical effects of exogenous OFQ on pain modulation. For example, OFQ produces a pronociceptive effect in the brain and an analgesic effect in the spinal cord. In order to better understand the physiological actions of OFQ, the present study focused on the pain-modulatory effect of endogenously released OFQ measured using antibody microinjection techniques. We found that electroacupuncture analgesia (EA) was increased by intracerebroventricular (i.c.v.) injection of an OFQ-antibody and decreased following intrathecal injection. Furthermore, i.c.v. OFQ-antibody partially reversed tolerance to both chronic morphine and chronic EA. These data suggest that endogenously released OFQ plays an important role in pain modulation, where pain sensitivity in the brain and spinal cord is increased and decreased, respectively.     

Increased conformational rigidity of humic substances by oxidative biomimetic catalysis

A synthetic water-soluble meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(III) chloride, Fe(TDCPPS)Cl, was employed as a biomimetic catalyst in the oxidative coupling of terrestrial humic materials. High-performance size-exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance (CPMAS-(13)C NMR), electron paramagnetic resonance (EPR), and diffuse reflectance infrared spectroscopy (DRIFT) were used to follow conformational and structural changes brought about in different humic materials by the oxidative coupling. Increase in apparent weight-average molecular weight (Mw(a)) occurred invariably for all humic substances with the oxidative polymerization catalyzed by Fe(TDCPPS)Cl. HPSEC further showed that the polymerization reaction turned the loosely bound humic supramolecular structures into more stable conformations which could no longer be disrupted by the disaggregating effect of acetic acid. DRIFT spectroscopy suggested the formation of new alkyl and aromatic ethers following the oxidative coupling with the biomimetic catalyst. CPMAS-(13)C NMR and EPR spectra suggested a reduced molecular mobility of humic components and enhanced stabilization of free radicals in larger oxidized fragments. All findings concur in indicating that the biomimetic catalysis by Fe(TDCPPS)Cl increased the molecular mass and chemical rigidity of humic materials by formation of intermolecular covalent bonds via a free-radical mechanism. The development of a technology based on oxidative polymerization by biomimetic catalysis may be of importance in controlling the properties and reactivity of humic matter for industrial and environmental applications.     

Description of characteristics of humic substances from different waste materials

Humic acids (HAs) extracted from different organic wastes have been characterised by chemical methods. The chemical properties of HAs showed differences depending on the source from which they were obtained. The C content in HAs from organic wastes (41.1-63.2%) fluctuated around the C value in soil HA with the exception of composted bark and tobacco dust. Compared with soil HA, the N contents of HAs from sewage sludge and brewery sludge were found much higher than the others. E4:E6 ratios for HAs in organic wastes were generally greater than that for soil HA, which indicated a low degree of condensation and humification. The carboxyl and phenolic-OH group contents ranged 0.51-2.23 and 11.1-20.7 meq g(-1), respectively. High values of carboxyl and phenolic-OH contents indicated that these materials were still within early stages of humification.     

The influence of humic substances on lacustrine planktonic food chains

Humic substances (HS) might influence planktonic food chains in lakes in two ways: 1) by altering the physical or chemical environment and thus modifying autotrophic primary production and the dependent food chains; 2) by acting as a direct carbon/energy source for food chains. HS compete with phytoplankton for available quanta underwater and this effect is seen in the reduced euphotic zone depth in lakes with high concentrations of HS. Thus potential photosynthetic production is lower in the presence of HS. However, this effect can be offset in small lakes in which the depth of mixing is also reduced when HS concentrations are high. Complexation by HS of important nutrients such as iron and phosphorus may also restrict primary production. Evidence is accumulating that photosynthetic primary production is insufficient to support measured metabolic activity in humic lakes, which implies that metabolism of allochthonous HS underpins much of the observed activity. Studies of bacterial abundance and growth in the presence of HS support the view that bacteria are the most significant utilisers of HS. This use is apparently facilitated by photolysis of HS, particularly by short wavelength radiation. Bacteria are grazed by both micro-zooplankton (heterotrophic and mixotrophic flagellates and ciliates) and macrozooplankton. It is within this microbial community that the food chains derived from autotrophic and allotrophic sources interact. These effects of HS on food chains are discussed in relation to possible implications for the response of different lake types to eutrophication.