Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.

A gloverin-like antibacterial protein is synthesized in Helicoverpa armigera following bacterial challenge.

A bacteria inducible antibacterial protein, P2, was isolated from the old world bollworm Helicoverpa armigera. Fifth-instar larvae were injected with live Escherichia coli NCTC 8196. P2 was isolated by HPLC using reversed-phase and size-exclusion columns. In addition, P2 was isolated by an alternative method of sequential cation-exchange and reversed-phase HPLC. The structure of P2 was determined by N-terminal Edman degradation and mass spectrometry. P2 had similar mass (14.1 kDa) structure and activity to gloverin, an inducible glycine-rich antibacterial protein isolated from Hyalophora gloveri [Axén, A.; Carlsson, A.; Engström, A.; Bennich, H. Eur. J. Biochem. 247:614-619; 1997]. At the N-terminus P2 had approximately 60% identity with gloverin. P2 is basic, heat stable, and displayed rapid antibacterial action. P2 was active against the Gram-negative bacteria tested and was inactive against the Gram-positive bacteria, Candida albicans, a bovine turbinate cell line, and pestivirus.

Isolation from an ant Myrmecia gulosa of two inducible O-glycosylated proline-rich antibacterial peptides.

Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline ( approximately 30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.

Antimicrobial mode of action of secretions from the metapleural gland of Myrmecia gulosa (Australian bull ant).

Secretions from exocrine metapleural glands of Myrmecia gulosa (Australian bull ant) exhibit broad-spectrum antimicrobial activity. Treatment of the yeast Candida albicans with metapleural secretion resulted in the rapid and total leakage of K+ ions from cells within 10 min. Ultrastructural analysis of the bacteria Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa, and cells and protoplasts of Candida albicans demonstrated gross damage of the cell membrane and aggregation of the cytoplasmic matrix of treated cells. Degradation of membrane-bound organelles was also observed in Candida albicans. The antimicrobially active components of metapleural secretions were nonpolar and interacted with the phospholipid bilayer, causing damage to the structural integrity of liposomes and the release of carboxyfluorescein. The data suggest that the antimicrobial agents in metapleural secretion act primarily by disrupting the structure and function of the phospholipid bilayer of the cytoplasmic membrane.

Antimicrobial properties of secretions from the metapleural glands of Myrmecia gulosa (the Australian bull ant).

Myrmecia gulosa (Australian bull ant) produce secretions from their metapleural exocrine glands which have broad spectrum antimicrobial properties. Such secretions are probably of importance in disease control in bull ant communities. These antimicrobial secretions are stable at 100 degrees C, resistant to proteolytic enzymes and are active over a wide pH range. Of the organisms tested only endospores of Bacillus cereus were found to be resistant. The antimicrobial agent(s) are absorbed by cells and result in cell lysis. The secretions do not interfere with any growth-related processes. These observations demonstrate that insects may be a source of novel antimicrobial agent(s).

Mutualisms: Assessing the benefits to hosts and visitors.

A great number and variety of interactions are widely assumed to be mutualistic because the species involved exchange goods or services from which they appear to derive benefit. A familiar example is pollination, in which animal vectors receive food in the form of nectar and/or pollen, while the ovules of plants are fertilized. Unfortunately, most studies fail to demonstrate that both participants benefit in any significant way and therefore lack the information necessary to determine whether a given interaction is mutualistic. While mutualism is thought to be a common type of species interaction, there is still little evidence for this belief.

Computer linked apparatus for upper limb therapy: a new system of resistive exercise.

A need has been recognised for equipment that can be used to provide objective evaluation of exercise of the upper limb in occupational therapy while being motivational to the patient. The microcomputer was seen to be a suitable tool as part of a system that might satisfy this need. After consulting experienced hand therapists the criteria were established for equipment that would encourage and quantify exercise. Three systems were built that represented three methods of resistive exercise. These were presented to therapists in a structured assessment to determine the most suitable. As a result of this a system employing a dashpot and a potentiometer connected to a BBC microcomputer was designed. Software was written in the form of activities that could be controlled by the movements of exercise on the equipment. Dashpot resistance could be varied and graded and the range of software was of varied motivational content. This allowed the principal movements of the wrist and forearm to be objectively exercised. After suitably calibrating the system it was used to assess patient effort and ability, and hence progress of treatment.

Pseudocopulation of an orchid by male ants: a test of two hypotheses accounting for the rarity of ant pollination.

The orchid Leporella fimbriata is pollinated by pseudocopulation with winged males of the ant Myrmecia urens. This recently studied interaction provides a unique opportunity to examine the two current hypotheses concerning the apparent rarity of ant pollination systems worldwide. The first hypothesis requires a series of specialized growth forms and floral characteristics regarded as adaptations to ant pollination. L. fimbriata does not possess them. The second considers the pollenicidal effects of secretions from the metapleural gland of ants. These glands are absent in M. urens males and it may be that the occurrence of ant pollination requires the absence of metapleural glands in the vector.

Plant dispersion, pollination and gene flow in Viola.

The effect of pollinator activity on gene flow in colonies of Viola were examined by measuring pollinator flight distances, the frequency of interplant flights and percent pollination under different plant spacing patterns. Pollinator flight distances were directly proportional to spacing parameters while the frequency of interplant flights and percent pollination were inversely proportional to spacing parameters. These findings show that gene flow is reduced by pollinator activity over a wide range of spacing parameters but in populations with low spacing means highly localized gene exchange can occur within the colony. Isolation of colonies may be expected under these circumstaces and cleistogamy may be the optimal breeding system. However, chasmogamous flowers may be important both in promoting within-colony gene exchange and long distance between-colony gene exchange corresponding to the sexual functions proposed in several recent models. Viola colonies appear to be semi-isolated demes with pollinator service which can bring adaptive genes to high localized frequencies, but which maintains low frequency, long-distance gene dispersal. This pattern corresponds to the "Shifting Balance" view of evolution.