Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses.

INTRODUCTION: Metabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds. OBJECTIVE: We aimed to combine LC-MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics. METHODS: We developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity. RESULTS: Among the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel. CONCLUSION: Our results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC-MS.

Actin organization during eucalyptus root hair development and its response to fungal hypaphorine.

The fungus Pisolithus microcarpus establishes an ectomycorrhiza with Eucalyptus globulus. This symbiosis involves a fungal synthesis and secretion of hypaphorine, an indolic compound. Previous studies have shown that hypaphorine induces an alteration in the actin cytoskeleton of elongating root hairs and inhibits hair elongation. Using an alternative approved method, we analyzed the effects of hypaphorine on the E. globulus root hair cyto-architecture and actin configuration in more detail and provide new results. One mM hypaphorine stops root hair elongation within 20 min, and changes the hair cyto-architecture. Semi-quantitative analysis of the actin cytoskeleton before and after treatment with hypaphorine shows that hypaphorine induces a shift from fine F-actin to F-actin bundles in the sub-apex of the hair, which occurs first in the mid-plane of the cell. This creates a sub-apical cell centre free of filamentous actin, an actin configuration that differs from that during developmental growth arrest. The mechanism of action of hypaphorine is discussed.

Spectrin-like proteins in plant nuclei.

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220-240 kDa, typical for human erythrocyte spectrin, and a prominent 60 kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

Spectrin-like proteins in green algae (Desmidiaceae).

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.

Isozymes as biochemical and cytochemical markers in embryogenic callus cultures of maize (Zea mays L.).

Isozyme analyses were carried out on protein extracts of non-embryogenic and embryogenic callus fromZea mays L., using polyacrylamide gel electrophoresis. We examined the isozyme patterns of glutamate dehydrogenase, peroxidase and acid phosphatase for their utility as biochemical markers of maize embryogenic callus cultures. These isozyme systems were also used to examine possible correlations between isozymes and different stages of regeneration. The zymograms of peroxidase and glutamate dehydrogenase differed for non-embryogenic and embryogenic callus. Further, some isozymes were correlated with the morphological appearance of the tissue while others seemed to be involved with the duration of the culture period. Using the same enzyme assays on fresh tissue samples we were able to test the three enzymes as cytochemical markers in embryogenic cultures. Glutamate dehydrogenase proved to be most successful to discriminate embryogenic from non-embryogenic cells.