A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen

Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR↓STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors

Binding of monoclonal anti-myelin-associated glycoprotein antibodies to human foetal peripheral neurons in culture

Dorsal root ganglion cells, obtained from 8-10-week human foetuses, were isolated by enzymatic procedure and grown on poly-l-lysine-coated coverslips. Most of the cultured cells showed the ultrastructural and immunological features of normal peripheral neurons. By immunocytochemistry neurons reacted with IgM antibodies with specificity for myelin-associated glycoprotein (MAG) from patients affected with IgM k gammopathy and peripheral neuropathy. The antigen was located on the plasmalemma of both perikarion and axon. We suggest that anti-MAG antibodies do not recognize neuronal MAG, but rather an epitope shared with different glycoproteins. \ua9 1987 Springer-Verlag

Effetto del deficit idrico sull’attività degli enzimi antiossidanti e sul trasporto elettronico fotosintetico in frumento duro.

VITERB

The maintenance of photosynthetic electron transport in relation to osmotic adjustment in durum wheat cultivars differing in drought resistance

The relationships between photosynthesis and osmotic adjustment (OA) under water stress are not yet well understood. Therefore, a possible association between the quantum yield of photosynthetic electron transport at the Photosystem II level (Φ(PSII)) and OA was evaluated in drought-stressed durum wheat plants in this paper. Four cultivars (cvs) differing in the degree of drought resistance as estimated by the Φ(PSII) maintenance, were grown in a growth chamber under two different water stress cycles; in both cases two stress intensities (moderate and severe) were used. Leaf water potential (Ψ(W)), osmotic potential at full turgor(Ψ(π100)), Φ(PSII) and the efficiency of excitation capture by open Photosystem II reaction centres (EC(PSII)) were determined in the different experimental conditions, both in control and stressed plants. Φ(PSII) and EC(PSII) were evaluated by in vivo chlorophyll fluorescence analysis. Φ(PSII) showed a significant decrease only under severe water stress and for the drought susceptible cvs; on the other hand, EC(PSII) showed no change under the different water stress conditions. The percent decrease of Φ(PSII) under water deficit was confirmed to be a good indicator of drought resistance in durum wheat. OA was estimated as the difference in the Ψ(π100) between leaves from water- stressed and well-watered plants. No relationship between the maintenance of the Φ(PSII) and OA under water deficit was observed in the adopted experimental conditions; this result is discussed in the light of the contrasting evidence in the literature