3. Key aspects of Fungiflex research
Let’s start with an overall summary of the key aspects of this research.
Crude extracts from Coprinopsis cinerea stipes and caps were found to have growth factor-like activities in standardised vertical stipe bioassays. Importantly, only the gentlest extraction technique (infusion in distilled water) was used, thus avoiding release of the cytosolic components which may have degraded growth factor activity and/or grossly contaminated the extract with other irrelevant components.
The activities observed, one with ‘contractile properties’ named Fungiflex 1, and the other with ‘extension-stimulating properties’ called Fungiflex 2, were attributed to two different substances, which were eventually found to be physically and chemically separable and therefore distinct, though possibly related compounds.
Microscopic analysis of their effects on vertical stipes, like those used in the standard bioassays, revealed that the substances penetrated tissue laterally very quickly since hyphal shape changes were apparent after only a 10-minute exposure. Yet, their activities were separated significantly in time, with the stipe-bending effects of Fungiflex 1 observed first, and being evident after 1 h, and the effects of Fungiflex 2 apparent some hours later.
Fungiflex 1 caused hyphae in the stipe to become flaccid and produce a large amount of extracellular material, which might be an indication of a role in permeabilising the plasma membrane. However, this morphology would also be consistent with signs of cell stress; application of Fungiflex 1 in a highly concentrated form probably caused many hyphae to collapse and eject their cytoplasmic contents. This was sometimes an inevitable pitfall of working with impure extracts in which the actual amount of applied growth factor was unknown.
Fungiflex 2 activity resulted in enhanced extension and not expansion of hyphae. Confirmation of the roles of Fungiflex 1 and 2 as growth factors responsible for hyphal inhibition and enhanced hyphal extension, respectively, requires further microscopic investigation of longitudinal stipe sections.
The specificity of the growth factor-like responses was confirmed when the two components responsible, Fungiflex 1 and Fungiflex 2, were separated by Thin Layer Chromatography (TLC) and their separate activities and molecular weights as well as spectral (IR) properties confirmed.
Similarity of the bending activities of some inorganic acids, bases and salts, as well as some amino acids to that of Fungiflex 1 was a coincidental outcome of the high concentrations that had to be applied causing an osmotic shock or a pH imbalance, rather than specific inhibition of hyphal elongation. Enhanced growth was not observed when stipes were treated with low concentrations of these inorganic compounds and amino acids. No bending activity was observed in stipes treated with various concentrations of mono- and disaccharides either.
Whereas many compounds elicited contraction, albeit at elevated concentration, significantly fewer exhibited Fungiflex 2-type activity: among those tested, just indole-3-acetic acid (IAA), ammonium formate and possibly cAMP.
Fungiflex 2 was determined to be different from these compounds by virtue of its greater molecular weight and its UV/visual and IR spectral patterns. However, the fact that these compounds, as well as the nicotinamide and/or ADP-ribose degradation products of NADase digestion of β-NAD, exhibited similar activity to Fungiflex 2 suggested that they may well all share chemical/functional groups which might have been at least partially responsible for the manner in which these substances activated enhanced extension; for example, ammonium formate and Fungiflex 2 shared carboxylic acid moieties.
Fungiflex activity was developmentally-regulated. The fact that Fungiflex 1 was found in extracts of all the developmental stages of Coprinopsis cinerea examined suggests that it was a constituent of multihyphal C. cinerea structures. Its production was possibly correlated with multihyphal development, although vegetative, dikaryotic mycelia were not analysed. Its activity was similar to the primordial cap inhibitor(s) extracted from Coprinus congregatus (= Coprinellus congregatus) (Robert & Bret, 1987; Robert, 1990), shown to be involved during the photoinhibition phases of mushroom fruit body development.
Copyright © David Moore & Lily Novak-Frazer 2016