Raman spectroscopic monitoring of Lactarius latex

Lactarius is a genus of Basidiomycotina with mainly agaricoid representatives, which are characterised by the excretion of a typical milky fluid. In particular, the colour, changes and taste of this latex-like milk are often used as a taxonomically important character. When it is exuded several chemical reactions occur. To date, NMR spectroscopy is generally used for chemical investigation of this latex. However, as a vibrational spectroscopic technique Raman spectroscopy has several advantageous properties for this type of research. The aim of this study is to investigate whether Raman spectroscopy can be used as an alternative analytical technique to monitor the chemical reactions in Lactarius latex. Therefore, this paper presents the first Raman spectra of Lactarius latex and provides an interpretation of the Raman bands that are present. L. lacunarum latex spectra are thoroughly investigated by 2D correlation analysis and are compared with latex spectra of other species (L. chrysorrheus, L. deterrimus, L. fluens, L. glyciosmus and L. salmonicolor).     

Short- and Long-term Tannin Induced Carbon, Nitrogen and Phosphorus Dynamics in Corsican Pine Litter

Pine litter amended with either tannic acid (TA) or condensed tannins (CTs) was studied to assess the effects on C, N and P mineralization in relation to the fate of tannins by incubation experiments during various time intervals. TA induced a rapid short-term effect resulting in high C respiration and net N and P immobilisation. After one week of incubation, TA was decomposed and net C, N and P mineralization and net nitrification resembled that of the control (non-amended litter). CTs exhibited effects on net mineralization on longer terms, i.e. after several weeks of incubation until the end of the experiment (84 days). While net N and P mineralization were greatly reduced, net nitrification was only slightly affected. Most likely CTs formed complexes with organic N of the substrate thereby reducing net N mineralization, while such complexes were not involved in net nitrification processes. The reduction of net P mineralization is due to the lack of need for P by microbes when they cannot get access to N. The fact that decreasing amounts of extractable CTs were accompanied by increasing effects on mineralization processes with incubation time strongly suggests that CTs were incorporated into the litter in such a way that they were inextricable by the common solvents needed to measure tannins, such as for the Folin–Ciocalteu and HCl–butanol assays.     

Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations

In most natural environments as well as in engineered environments, such as wastewater treatment plants, ammonia-oxidizing bacteria (AOB) experience fluctuating substrate concentrations. Several physiological traits, such as low maintenance energy demand and decay rate, cell-to-cell communication, cell mobility, stable enzymes and RNAs, could allow AOB to maintain themselves under unfavourable circumstances. This review examines whether AOB possess such traits and how these traits might offer advantages over competing organisms such as heterotrophic bacteria during periods of starvation. In addition, within the AOB groups, differences exist in adaptation to and competitiveness under conditions of high or low ammonia or oxygen concentrations. Because these findings are of importance with regard to the ecology and activity of AOB in natural and engineered environments, concluding remarks are directed towards future research objectives that may clarify unanswered questions, thereby contributing to the general knowledge of the ecology and activity of ammonia oxidizers.     

Modelling genetic regulation of growth and form in a branching sponge

We present a mathematical model of the genetic regulation controlling skeletogenesis and the influence of the physical environment on a branching sponge with accretive growth (e.g. Haliclona oculata or Lubomirskia baikalensis). From previous work, it is known that high concentrations of silicate induce spicule formation and upregulate the silicatein gene. The upregulation of this gene activates locally the production of spicules in the sponge and the deposition of the skeleton. Furthermore, it is known that the expression of the gene Iroquois induces the formation of an aquiferous system, consisting of exhalant and inhalant pores. We propose a model of the regulatory network controlling the separation in time and space of the skeletogenesis and the formation of the aquiferous system. The regulatory network is closely linked with environmental influences. In building a skeleton, silicate is absorbed from the environment. In our model, silicate is transported by diffusion through the environment and absorbed at the surface of a geometric model of the sponge, resulting in silicate gradients emerging in the neighbourhood of the sponge. Our model simulations predict sponge morphology and the positioning of the exhalant pores over the surface of the sponge.     

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Reconstruction of protein backbones from the BriX collection of canonical protein fragments

As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures.     

A simple method to vary soil heterogeneity in three dimensions in experimental mesocosms

Soil heterogeneity affects terrestrial plant communities both directly and indirectly. In nature, the exploration of the role of heterogeneity is made difficult because any co-varying factors (nutrients, soil depth, etc.) render it problematic to clearly link cause and effect. Attributing changes specifically to heterogeneity is facilitated if heterogeneity is varied in a controlled manner and other possible confounding factors are kept constant. The experiments conducted in such a way have up till now only considered heterogeneity in two dimensions, horizontally or vertically. In this methodological study, we present a novel technique that enables researchers to vary both qualitative and configurational heterogeneity in three dimensions by building up the soil cell by cell in experimental mesocosms. We illustrate the technique with an experiment where we test the effect of cell size (i.e. configurational heterogeneity) on the performance of grassland species that vary in nutrient preference (high N and low N species). Cell size did not affect aboveground biomass but modified species richness, both at the mesocosm and the patch scale, with most species being found when cells were small yet distinct (cell size 12 cm). High N species had significantly greater aboveground biomass and species richness than low N species, both on nutrient rich and nutrient poor cells. Remarkably, those differences disappeared when plants grew on the mesocosms with cell size close to zero. By allowing greater complexity in the design of experimental mesocosms, the 3-D approach can improve understanding of the interplay between soil heterogeneity and plant and ecosystem functioning.     

Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies

We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies. Received: 26 February 2000 / Accepted: 26 January 2001     

Effect of nitrogen concentration on fructan and fructan metabolizing enzymes in young chicory plants (Cichorium intybus)

Witloof chicory seeds ( Cichorium intybus L. var. foliosum cv. Flash) were sown in acid-washed vermiculite in a controlled environment growth chamber. Plants received a nitrogen poor ("N-poor": 0.2 m M NH₄NO₃) but otherwise complete medium, or a nitrogen rich ("N-rich": 2 m M NH₄NO₃) medium. After 1 month of growth the fructan concentration in the "N-poor" plants was about five times higher and also the activity of sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) was twice as high as in "N-rich" plants. The activities of the catabolic enzymes fructan 1-exohydrolase (1-FEH; EC 3.2.1.80) and acid invertase (EC 3.2.1.26) were higher in the "N-rich" plants where significant energy was invested in root and leaf growth. After one month of growth, part of the "N-poor" plants were switched to the "N-rich" medium. One day after this switch, a sharp decrease in sucrose and glucose concentration was observed in the roots. During the following days, both the activities of 1-SST and fructan:fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) decreased and the 1-FEH and invertase activities increased. These changes were correlated with a decrease in fructan concentration. Ten days after the switch, glucose and sucrose concentrations increased again and fructan synthesis resumed. During this period 1-SST activity increased and 1-FEH activity decreased. Apparently 1-SST, 1-FFT and 1-FEH simultaneously control fructan in young chicory roots. The rather unexpected finding that 1-FEH activity, which was believed to occur only in older material, can be induced in very young roots indicates that this enzyme can be induced at any physiological stage.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).