Geoffrey R. Moores

Micropatterned substratum adhesiveness: a model for morphogenetic cues controlling cell behavior.

It is generally considered that tracks of cell adhesiveness are important in controlling cell migration during the development and regeneration of many tissues. In order to investigate this experimentally, a number of techniques have in the past been employed to make patterns of differential adhesiveness for in vitro studies. However, practical limitations on patterning resolution and the introduction of residual topography to the experimental substrata have restricted their usefulness. Here we describe a simplified photolithographic technique for patterning cell adhesiveness which allows a high degree of flexibility and precision. We have quantified, using adhesion and spreading characteristics of BHK cells, the differential adhesiveness that can be created on patterned surfaces, how this alters with the duration of exposure to serum proteins, and how this, in turn, relates to the persistence of cell patterning despite increases in cell density. We believe that this technique will prove extremely useful for the detailed in vitro examination of the mechanisms controlling cell behavior as it offers a degree of precision and ease of fabrication that has previously been unavailable.

Microelectronic and Nanoelectronic Interfacing Techniques for Biological Systems

A new field of research and development has emerged in recent years which relies on the availability of ultra-high precision technology and cross-disciplinary collaboration between electronic engineers, biologists, biochemists and chemists. By exploiting the techniques of photolithography and electron beam lithography devices can be created to interface with biological cells or molecules such as enzymes. This type of work is increasingly referred to under the heading of ‘bioelectronics’ and touches on topics such as biosensors and cell-electrode interfacing.

Conductimetric assay of phospholipids and phospholipase A

Direct measurement of chemical change during enzyme-catalysed hydrolysis is, in principle, an attractive method for qualitative and quantitative analysis of phosphatidyl phospholipids. Phospholipase type AZ (EC 3.1.1.4) of high activity and broad specificity is available from bee venom, the source used in this work, and from mammalian pancreas. We have developed a rapid conductimetric assay which has a detection level below 10 pg of phospholipid (l-2 pg Pi) and below 10 ng of purified enzyme. The reaction: lecithin -+lysolecithin + fatty acid anion -+ H+ has been followed by titration [l] or by turbidity change in protein-rich solution [2], but neither of these methods has been adapted for routine estimation of phospholipids. The conductimetric method [3,4] requires minimum sample preparation and the apparatus is simple and readily adapted for routine largescale determinations. Bee venom enzyme has highest activity with substrates in true solution [l] , and for naturally occurring phospholipids this is obtained in dilute aqueous solutions of organic solvents; however, the intrinsic activity of the enzyme is decreased by organic solvents and optimum rates are given in 12-2% n-propanol : water mixtures. In addition activity is increased 5-lO-fold by 0.1 mM Ca2+ but abolished by excess EDTA [ l] .

Activation of bee venom phospholipase A2 by fatty acids, aliphatic anhydrides and glutaraldehyde.

The phospholipase from Bee venom, a small cationic protein (mol. wt 14 629, p1 10.5 ? 1.0) of known primary structure [ 1,2] hydrolyses long-chain phosphatidyl phospholipids most rapidly in dilute aqueous solutions of organic solvents (e.g. n-propanol) where it shows’product activation [3] which becomes more pronounced as the solvent concentration is decreased. Activation of lipases is a common phenomenon of possible importance in many regulatory processes [4,5] and the explanations proposed concentrate on possible modification of the substrate by the activator to increase attraction to, or penetration of the surface by the enzyme [6,7]. The Bee venom enzyme is activated by fatty acid anions (e.g. palmitate, oleate) with a minor contribution from lysolecithin, fig. la, and both products are inhibitory although this is masked at low concentrations. The kinetics of the system are deceptively simple, an individual progress curve having a near linear slow phase followed after a brief transition by a fast, activated phase. Whilst the rate in the activated phase is highly dependent on substrate concentration, that in the slow phase is not, showing that activation, which increases V,,, approx. 25fold appears to decrease affinity for the substrate. This result is more readily explained (given in detail in a later paper) by postulating that activators modify the enzyme not the substrate, a conclusion strongly reinforced by the experiments presented here.

The preparation of activated bee venom phospholipase A2.

Many phospholipase enzymes are activated by long-chain fatty acids, but the sensitivity to activation depends on the nature of the substrate. It therefore has been concluded that fatty acids intercalate into the lipid phase and modify the primary interaction of the enzyme with its substrate [l] . Bee venom phospholipase A* is activated by fatty acids [2] , but the kinetics of activation do not support this substratemediated mechanism (unpublished data); however the high affinity of both substrate and enzyme for free long-chain fatty acids makes investigation by kinetic means both difficult and unreliable. This enzyme can be activated irreversibly by treatment with acid anhydrides presumed to add longchain acyl residues to nucleophilic groups in the protein [2]. If all of the effects of fatty acid activation were produced by covalent addition of a single acyl residue, fatty acid activation could be attributed to allosteric properties of the enzyme. It would then be feasible to design reagents for selectively activating or blocking the activation of this and related enzymes in vivo. Lauroyl ethyl carbonate (the mixed anhydride of lauric acid and ethyl carbonic acid) was chosen as activator for this study because it gave rapid activation with little non-specific inactivation (tested against a non-activating substrate) and free lauric acid is a relatively weak activator at corresponding concentrations. Dimethyl maleic anhydride, a reversible blocking agent for amino-groups [3] was used initially to protect the protein against non-specific acylation, but its direct action on fatty acid activation became of greater interest here.

Scanning force microscopy of protein-patterns

The development of microminiaturized biosensors requires techniques for immobilizing biomolecules on solid substrates, in an ordered fashion, and techniques for the subsequent visualization of these patterns. Scanning force microscopy (SFM) is a useful technique for visualizing ordered patterns, but it requires suitable substrates and attachment techniques. Here we present a photolithographic method which gives ordered patterns of biomolecules. Both SFM topographic and lateral force images of these patterns are shown and discussed.

High-resolution two-part basic urea gels for analysis of venom phospholipase A2 isoforms

The performance of acidic and basic urea polyacrylamide gels has been improved by adopting a two-part gel system with a concentration discontinuity to act as a stacking boundary and by increasing the urea concentration to 8 M. The contributions of primary amino-group and guanidino-group ionizations to mobility have been evaluated by acetylation and phenyl glyoxal treatment respectively. The chromogenic PLA2 detection method of Shier and Trotter (Analyt. Biochem. 87, 604, 1978) has been modified for use with basic urea PAGE. The results have confirmed the major findings of other workers, but have demonstrated the presence of many hitherto uncharacterised isoforms of PLA2 in a variety of whole snake venoms. The basic urea PAGE (BG) method is proposed as the basis of a simple and rapid method for the classification of PLA2 isoforms which should allow unambiguous identification of isoforms by referring bands for purified material to the isoform content of whole venoms.