TIBTECH- FEBRUARY1990[Vol. 8]
53
Protein-based medical adhesives R o b e r t L. S t r a u s b e r g and Rebecca P. Link
There are m a n y naturally occurring adhesive proteins w h i c h have potential for application in medicine and dentistry. Cloning and expression of their genes enables the modes of action of these proteins to be better understood and increases their availability for practical applications. This article concentrates on the adhesive protein from the blue mussel Mytilus edulis but also describes medical adhesives based on fibrin isolated from h u m a n blood. For many years adhesive chemists have been intrigued by the task of developing adhesive materials for in vivo medical applications in areas such as ophthalmology, plastic surgery, otology, cardiology, and neurology and orthopedics. Any adhesive material targeted for medical applications should possess the following general characteristics: • biocompatibility (non-toxic, low immunogenicity); • adherence and curing in moist environments; • non-interference with the natural healing process; • appropriate adhesive and cohesive properties.
considered as a natural adhesive for reuniting severed nerves 3'4. The use of fibrin sealants has increased dramatically in recent years (Box 1). For many applications, this material may be the adhesive of choice. However, fibrin adhesives are unlikely to have universal appeal in medical applications because the strength of the material (both adhesive and cohesive) is not adequate for many procedures, and also because fibrin sealants are derived from human blood (Box 1), and therefore might potentially carry viral contaminants 1°'17'18. In the United States, fibrin adhesive is prepared on small scale from the patient's own blood 1°'17'18, therefore eliminating
Researchers have focused on the use of natural proteins with adhesive properties• In this article, we discuss the genetic engineering and processing of adhesive proteins naturally produced by the mussel Mytilus edulis. We will also discuss, as a reference point, fibrin sealant systems: the greater depth of experience with the fibrin systems will illustrate the requirements and potential for other medical adhesives•
potential problems from contamination. However, this is a time consuming and costly approach. Therefore, the human origin of materials in fibrin sealants, a strong positive factor in biocompatibility, might also limit its use. While it would be desirable to genetically engineer components of the fibrin adhesive system to reduce the potential for contamination, the complex nature of fibrin would make this extremely difficult to achieve. This creates opportunities for other medical adhesives•
Adhesive materials synthesized by marine organisms Marine organisms including mussels and barnacles produce remarkable moisture-resistant adhesives. Since these are applied and cured in moist environments, they are potentially useful for many medical and dental applications. The blue mussel Mytilus edulis synthesizes a specialized polyphenolic adhesive protein which plays a key role in attachment to surfaces. The protein is located in a specialized thread-like structure called the byssus which also contains several other proteins including collagen. When secreted and applied, the byssal adhesive is highly cross-
:6
Fibrin sealant systems Fibrin sealant was the first proteinbased adhesive system marketed for in vivo applications. Fibrin, a key component of the natural hemostatic process, was first employed in surgical procedures during World War 11'2. Interest in this material revived during World War II when it was
I
"It's Robert L. Strausberg and Rebecca P. Link are at Genex Corporation, 16020 Industrial Drive, Gaithersburg, MD 20877, USA. @ 1990, Elsevier Science Publishers Ltd (UK)
0167 - 9430/90/$2.00
an a m b i t i o u s of the r e w a r d s
project
if w e ' r e
all r i g h t ,
but think
successful!"
TIBTECH - FEBRUARY 1990 [Vol. 8]
54
--Box I
The biochemistry of fibrin sealants and their application Since the Second World War, researchers have gained great insight into the biochemistry of the fibrinolytic and homeostatic systems. This has lead to the practical development of fibrin sealants containing purified protein components 5'6. These fibrin adhesives, currently marketed in Europe, are sold under tradenames such as Tissucol ®, Tisseel ® and Fibrin-Kleber Human Immuno ®. Fibrin sealant systems are currently being tested in diverse medical applications including nerve 7 and vascular a repair, opththalmic surgeries 9, cardiovascular grafts 1°, cartilage/bone grafting 11 and skin graft adhesion 12. Fibrin adhesive is generally used as a two component system. Component 1 contains at least 70 mg fibrinogen, 2-7 mg fibronectin, 10 units of Factor XIII, and 35 Fg of plasminogen and aprotinin in a l m l solution. The component is prepared by adding a solution of aprotinin to a lyophilized powder of the human proteins. Component 2 is a thrombin solution, prepared by adding a CaCI2 solution to lyophilized bovine thrombin. The time for the fibrin adhesive to set is control led by the amount of thrombin used in the formulation. The fibrinogen solution is viscous and upon mixing with the thrombin solution quickly sets to a firm, white gel. When this reaction occurs at the application site the sealant adheres to the tissue 5. The major protein in the fibrin adhesive is fibrinogen. This protein, with a molecular mass of 340 kDa, consists of six polypeptide chains (three pairs of nonidentical chains designated ~, [3 and 7) linked through disulfide
linked and cannot readily be analysed biochemically. However, substantial progress has been made in characterizing the non-cross-linked adhesive protein which is produced and stored in the phenol gland 19 21. The 130 kDa protein is rich in proline/ hydroxyproline, serine, threonine, lysine, tyrosine/3,4-dihydroxyphenylalanine (Dopa) and alanine, a composition which suits the protein for interaction with biological surfaces (Table 1). The protein is extremely hydroxyl-rich with 60-70% of the amino acid residues containing hydroxyl groups. A high proportion of its proline residues are post-translationally hydroxylated to either 3- or 4-hydroxyproline 2°. In addition, more than half of its tyrosine residues are converted to Dopa. The decapeptide sequence Ala-Lys-Pro/HypSer-Tyr/Dopa-Hyp-Hyp-Thr-DopaLys was found by peptide sequence analysis to occur repeatedly 21. Recently, formulations of the natural mussel adhesive protein (MAP) have been tested in ophthalmic applications 22'23 and the preliminary indications are that the adhesive has potential both in the treatment of corneal perforations and in epikeratoplasty.
bridges. The proteolytic action of thrombin releases peptides from the amino termini of the o~ and [3 chains, thereby stimulating spontaneous polymerization of the fibrin monomers. At this point in the reaction, the fibrin monomers are not joined by strong covalent bonds. However, thrombin in the presence of Ca 2+ also catalyses the activation of the zymogen, Factor XIII, to the transaminase, Factor XIIla. The Factor XIIla catalyses formation of stable 7-glutamyl-e-lysine cross-links, adding to the strength and stability of the fibrin clot. This in vitro process mimics the natural reaction that results in the formation of a fibrin clot. Details of the fibrinogenfibrin system have been reviewed 13. Other components in the fibrin sealant are included to aid in the adhesion process. Fibronectin, a natural cellular adhesive, enhances the adhesion of fibrin to tissue, possibly through cross-links to the major vertebrate structural protein, collagen 14,15. Aprotinin and plasminogen are not essential for the formation of fibrin clot but are included in fibrin sealant formulations to control fibrin biodegradability. Aprotinin suppresses fibrin degradation. The natural protease for fibrin is plasmin, activated by tissue plasminogen activator TM. Inclusion of an inhibitor like aprotinin slows the degradation process to allow for natural healing. The fibrin sealant does not interfere with natural healing and is eventually degraded. Plasminogen, a component of the fibrin sealant, assists in the complete degradation of the adhesive.
Cloned DNA encoding the mussel adhesive protein In order to further investigate its amino acid sequence and to develop a microbial system for expression of the mussel adhesive protein, the Genex research group prepared cDNA libraries from Mytilus edulis phenol gland mRNA 24 and identified sequences encoding portions of the adhesive protein. Analysis of these clones revealed that the mussel adhesive protein carries a family of related decapeptide and hexapeptide sequences arranged as tandem repeats. In general, the decapeptide raTable I
Potential adhesive interactions of frequently occurring amino acids in M y t i l u s e d u l i s adhesive protein A m i n o acid
C h e m i c a l bond
Serine Threonine Lysine Tyrosine Hydroxyproline Dopa
Hydrogen Hydrogen Ionic Hydrogen Hydrogen Metal complex formation
repeat units have the formula A-LysB-Ser/Thr-Tyr-Pro-Pro-Thr/Ser-TyrLys, where A is usually Ala or Pro and B is usually Pro. However, many sequence variations are observed. The most highly conserved residues in the various decapeptide repeat units are tyrosine and lysine, and proline at position 6. The DNA sequence analysis 24 confirmed the repetitive nature of the adhesive protein and revealed many related decapeptides and hexapeptides not previously identified by peptide sequencing 21. Microbial production of mussel adhesive protein In order to produce sufficient quantities of adhesive protein for studying adhesive mechanisms and to develop materials for medical and dental applications, Genex scientists have developed a microbial system to produce the protein 25. Although microbial expression of the mussel gene might be expected to be similar in many respects to the expression of other higher eukaryotic genes, some particular considerations were made. Firstly, because the protein is encoded by repetitive DNA, it had to be established that the coding material would be stable in the microbial host.
TIBTECH - FEBRUARY 1990 [Vol. 8]
55
--Fig. 1 2Ft L
: ~ G A L 1
highly expressed E. coli or yeast genes. However, our results 25 suggest that microbial cells can express foreign repetitive sequences well.
UAS
'~
//F-o~1TIS
PH05 signal
Hydroxylation ~D adhesive cDNA encoding protein
Am R
H terminator
Yeast vector for expression of mussel adhesive protein gene. This yeastE. coli shuttle vector is designed to direct high-level expression of foreign genes 25 It carries the yeast LEU2d gene for selection in leu2 yeast hosts and a
replication origin from the natural yeast 2# plasmid. Expression of the adhesive protein is regulated by a hybrid promoter carrying the upstream activation site (UAS) from the GAL 1 gene and the transcription initiation site (TIS) from the alpha-factor (MF-~I) gene. Transcription is terminated by a yeast glyceraldehyde-3-phosphate dehydrogenase terminator. Translation is initiated by a synthetic sequence carrying preferred yeast codons and encoding the yeast PH05 signal.
A yeast expression vector for the adhesive protein gene (Fig. 1) has been used to produce derivatives of the natural mussel adhesive protein ranging in molecular mass from 20 to 100 kDa at about 2-5% of total cell protein. Somewhat surprisingly, the highly repetitive coding sequences are very stable in yeast, even when carried on high copy number plasmids. Furthermore, we anticipated that production of the adhesive protein in E. coli or yeast might be limited because the natural repetitive coding sequences include codons which are not normally preferred in
Secondly, three amino acids, proline, tyrosine and lysine, comprise about 70% of the amino acids in the protein; therefore, efficient microbial expression was likely to be challenging. Thirdly, the need for post-translational hydroxylation would need to be addressed. The expression system developed at Genex was based on yeast. In general, the most efficient expression modules include a very well regulated promoter, a highly expressed yeast coding sequence fused to the repetitive gene and a transcription terminator.
--Fig. 2 OH
OH
Tyrosinase '& 02
OH2 I NH--CH--CO--
Tyrosine
0
Catechol oxidase .~
.,~ CH 2
I NH--CH--CO--
Dopa
7, 02 CH2
I NH--CH--CO--
o-Quinone
Post-translational modifications of tyrosine residues in the Mytilus edulis adhesive protein. Both the hydroxylation of tyrosine to Dopa and oxidation of Dopa to quinone can be catalysed by tyrosinases derived from mushrooms and bacteria.
The adhesive protein produced in yeast contains neither Dope nor hydroxyproline residues. The importance of hydroxyproline in byssal thread formation and adhesion is unknown. However, it has been suggested that hydroxyproline residues provide additional hydroxylgroups without losing the 'structure breaking' characteristics of proline. An alternative explanation might be that hydroxyproline residues are involved in stabilizing interactions between protein molecules in the byssal thread. Hydroxyproline residues do stabilize the triple helix of collagen 26. Collagen is a prominent protein in the mussel byssal thread 27 and may interact closely with the mussel adhesive protein. Dopa residues are quite unusual in proteins; it has been suggested that these residues may be crucial for moisture-resistant adhesion to surfaces and for cross-linking to build cohesive strength. Our studies with the genetically engineered adhesive protein support this; they show that cross-linking and moisture-resistant adhesion requires the presence of the reactive, oxidized form of Dope, quinone. To achieve moisture-resistant adhesion, there is a need for tyrosine residues to be hydroxylated. Fortunately, the post-translational modification of tyrosine to Dope and Dope to quinone can be catalysed in vitro by mushroom and bacterial tyrosinases (Fig. 2). Using a bacterial tyrosinase we have developed methods for in vitro production of the Dope-adhesive protein. The protocol is a modification of the procedures using mushroom tyrosinase described by Marumo and Waite 28. Ascorbic acid is added to reduce any quinone produced back to Dopa. The process produces an adhesive protein in which >50% of the tyrosine residues are converted to Dope. The Dope-adhesive protein is stable in an acidic solution or as a lyophilized powder. The Dope-protein is the preadhesive form of the protein. The adhesive properties are generated when the Dope residues are oxidized to quinone.
56
TIBTECH - FEBRUARY 1990 [Vol. 8]
--Fig. 3 a
Adhesive and cohesive properties The adherence of the engineered mussel adhesive protein in an aqueous environment to various surfaces including polystyrene, glass, hydrogel and collagen has been tested. In each case, activation to the quinone form of the protein was required for good surface adhesion. Adhesive protein bound to polystyrene was resistant to removal by washing with 0.9% NaC1, 0.5% SDS or Triton X-100, 1% acetic acid, 0.1 N NaOH, at 37°C, and sonication in water for 30 minutes. The cohesive strength of the engineered adhesive protein depends upon intermolecular cross-linking which requires formation of quinone residues. The nature of the chemical bond involved in cross-linking has not yet been determined, although it has been suggested that quinone residues could bond to the c-amino group of lysine through a Michael addition reaction. Quinone formation (and cross-linking ) can be achieved by either chemical or enzymatic oxidation of the Dopa residues. The enzymatic process permits regulation of cross-linking by altering the tyrosinase concentration. Importantly, certain formulations of the adhesive protein form gels following cross-linking that are similar in appearance to collagen and fibrin gels. Preliminary evidence suggests that cross-linking with other proteins will occur when these are present at the time of activation. The adhesive protein appears to have affinity for both soluble and insoluble collagen. Cross-linking the engineered adhesive protein with natural extracellular matrix proteins might enhance in vivo adhesive performance. In an in vitro model system in which moist collagen sheets were used as the substrate, the performance of the engineered adhesive was compared with that of fibrin adhesive. It was concluded that the engineered adhesive protein consistently had significantly greater strength than fibrin adhesive. The performance of the adhesive in vivo has been examined using a w o u n d healing model system in animals. The initial in vivo studies demonstrated that useful adhesive properties can be observed in less than one minute, and that adhesive strength increased for several hours
More recently, direct biochemical analysis has revealed Dopa residues b in structural/adhesive proteins from several species. For example, the marine polychaete Phragmatopoma californica lives in protective tubes C ~ formed by a combination of natural debris and secretions from the worm: Jensen and Morse 3° suggest that the Dopa residues in this protein may act through quinone tanning to stabilize the complex and form adhesive bonds. The putative egg shell pree i:i:i:i:i:i:i:l cursor proteins from the vitelleria of the liver fluke Fasciola hepatica 31'32 provide further examples of Dopacontaining proteins. These proteins, which apparently contribute to the sturdiness of the egg shell, are similar g •::i:i:i:i:!i:i:i:!i:i:i:!i:i:i:i:!:i:i:!:iI:!:i:i:!:i:i:!:i:i:!:i:i:!:i:i:i:!:i:i:•i:i:!:i:i:i!:i:i:toi:i:i:!the •i:i:!:i:imussel :i:!i:• adhesive protein in that they carry Dopa residues within Genetic constructions for adrepetitive peptide sequences. hesive proteins: (a) cloned cDNA Genetic engineering technology encoding a portion of the M y t i l u s can provide great flexibility in the e d u l i s adhesive protein (the seproduction of proteins for adhesive quence encodes repeating decaapplications. We have assembled peptide/hexapeptide sequence); synthetic genes encoding adhesive (b-d) tandem repeats of cloned proteins carrying repeated peptides cDNA encoding increasingly higher molecular weight forms of designed to enhance adhesion to the adhesive protein; (e) hybrid particular surfaces (Fig. 3). It might sequence encoding a cell attachalso be possible to enhance the ability ment peptide linked to the enginof engineered adhesive proteins to eered adhesive protein; (f) hybrid interact with cells in vivo by creating protein carrying multiple cell fusion proteins which include cell attachment domains derived from attachment sequences from extrafibronectin or other cell matrix cellular matrix proteins such as proteins; (g) adhesive protein fibronectin, laminin and vitronectin. carrying repeated peptide sequences designed to enhance These hybrid coding sequences (e.g. adhesion to particular surfaces. Fig. 3) can be assembled from (The coding sequence is assemcombinations of cDNA and synthetic bled from synthetic DNA.) DNA. Through this technology and careful protein design, it is likely that a new generation of biocompatible adhesive materials could be develafter initial application. Wound heal- oped and, during the next decade, ing in the presence of the adhesive these materials could become key components in surgica] and inteappears normal. grated w o u n d management proOther adhesive proteins grams. Microbial synthesis might well be applied to other structural/adhesive Acknowledgements proteins with moisture-~'esistant The Genex research on engineered properties. While the presence of adhesive proteins has been supDopa residues in the mussel adhesive ported by Phase I and Phase II SBIR protein is unusual, it is certainly not grants from the National Institute of unique. Indeed, quinone tanning of Dental Research. We gratefully proteins to achieve moisture-resist- acknowledge the contributions of our ance and to retard biodegradability colleagues in the Genex bioadhesive has been suggested before: in 1950, research group. Brown 29 noted evidence of quinone tanning in various structural proteins References of invertebrates including the byssus 1 Grey, E. G. (1915) Surg. Gyneco]. of Mytilus edulis and the egg case of Obstet. 21,452-454 the liver fluke, Fasciola hepatica. 2 Harvey, S. C. (1916) Boston Med.
TIBTECH- FEBRUARY 1990 [Vol. 8]
Surg. J. 174,658-659 3 Young, J. Z. and Medawar, P. B. (1940) Lancet ii, 126-128 4 Tarlov, I. M., Denslow, C., Swarz, S. and Pineles, D. (1943) Arch. Surg. 47, 44-57 5 Redl, H., Schlag, G. and Dinges, H. P. (1985) in Biocompatibility of Tissue Analogs Vol. 1 (Williams, D. F., ed.), pp. 135-157, CRC Press 6 Ellis, D. A. F. and Pelausa, E. O. (1988) J. Otolaryngol. 17, 74-77 7 Cruz, N. I., Debs, N., and Fiol, R. E. (1986) Plast. Reconstr. Surg. 78, 369-373 8 Barton, B., Moore, E. E. and Pearce, W. H. (1986) J. Surg. Res. 40,510-513 9 Rostron, C. K., Brittain, G. P. H., Morton, D. B. and Rees, J. E. (1988) Arch. Ophthamol. 106, 1103-1106 10 Dresdale, A., Bowman, F. O., Jr, Malm, J. R. et al. (1985) Ann. Thorac. Surg. 40, 385-387 11 Gersdorff, M. C. H. and Robillard, T. A. J. (1985) Laryngoscope 95, 1278-1280 12 Vibe, P. and Pless, J. (1983) Scan. I. Plast. Reconstr. Surg. 17, 263-264 13 Doolittle, R. F. (1984) Annu. Rev.
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Biochem. 53, 195-229 14 Williams, E. C., Janmey, P. A., Johnson, R. B. and Mosher, D. F. (1983) J. Biol. Chem. 258, 5911-5914 15 Barry, E. L. R. and Mosher, D. F. (1988) J. Biol. Chem. 263, 10464-10469 16 Lijnen, H. R. and Collen, D. (1987) Ann. Biol. Clin. 45, 198-201 17 Epstein, G. H., Weisman, R. A., Zwillenberg, S. and Schreiber, A. D. (1986) Ann. Otol. Rhinol. Laryngol. 95, 4O-45 18 Siedentop, K. H., Harris, D. M., Ham, K. and Sanchez, B. (1986) Laryngoscope 96, 1062-1064 19 Waite, J. H. and Tanzer, M. L. (1981) Science 212, 1038-1040 20 Waite, J. H. (1983) J. Biol. Chem. 258, 2911-2915 21 Waite, J. H., Houseley, T. and Tanzer, M. L. (1985) Biochemistry 24, 5010-5015 22 Robin, J. B., Picciano, P., Kusleika, R. S., Salazar, J. and Benedict, C. (1988) Arch. Ophthalmol. 106, 973-977 23 Robin, J. B., Picciano, P. and Benedict, C. V. (1988) in The Cornea Trans-
24
25 26
27 28 29 30 31 32
actions of the World Congress on the Cornea lll (Cavanagh, H. D., ed.), pp. 55-58, Raven Press Stransberg, R. L., Anderson, D. M., Filpula, D. et al. (1989) in Adhesives from Renewable Resources: ACS Symposium Series 385 (Hemingway, R. W. and Conner, A. H., eds), pp. 453-464, American Chemical Society Strausberg, R. L. and Strausberg, S. L. (1988) PCT Patent Application No. US87/02663 Kivirikko, K. I. and Myllyla, R. (1980) in The Enzymology of Posttranslational Modification of Proteins (Freedman, R. B. and Hawkins, H. C., eds), pp. 53-104, Academic Press Mascolo, J. M. and Waite, J. H. (1986) J. Exp. Zool. 240, 1-7 Marumo, K. and Waite, J. H. (1986) Biochim. Biophys. Acta 872, 98-103 Brown, C. H. (1950) Nature 165, 275 Jensen, R. A. and Morse, D. E. (1988) J. Comp. Physiol. B. 158,317-324 Waite, J. H. and Rice-Ficht, A. C. (1987) Biochemistry 26, 7819-7825 Waite, J. H. and Rice-Ficht, A. C. (1989) Biochemistry 28, 6104-6110
A d d i t i o n a l i n f o r m a t i o n m a y be o b t a i n e d f r o m IBPGR H e a d q u a r t e r s , Via delle Sette Chiese 142, 00145 R o m e , Italy.
Genetic resource corrections We w o u l d like to p r o v i d e clarification on the t y p e of p l a n t genetic r e s o u r c e s i n f o r m a t i o n that is m a i n t a i n e d b y the I n t e r n a t i o n a l Board for Plant Genetic Resources (IBPGR). Most of the i n f o r m a t i o n p r e s e n t e d a b o u t IBPGR b y D. Jane B o w e r in her article ' G e n e t i c resources w o r l d w i d e ' (Ref. 1) was not accurate due to a shift in IBPGR p r o g r a m m e s a n d a misu n d e r s t a n d i n g in t e r m i n o l o g y . IBPGR does c o o r d i n a t e an intern a t i o n a l s y s t e m of p l a n t genetic r e s o u r c e centres. Both n a t i o n a l a n d i n t e r n a t i o n a l institutes participate. IBPGR p r o m o t e s seed genetic resource conservation by providing financial a s s i s t a n c e a n d scientific expertise, but the m a n a g e m e n t of the collections is p e r f o r m e d b y each institute. Currently, h o w e v e r , IBPGR is not d e v e l o p i n g a n e t w o r k of active collections; rather it is facilitating the e s t a b l i s h m e n t of crop networks. T h e s e will e n c o m p a s s all interested parties i n v o l v e d w i t h an i n d i v i d u a l c r o p i n c l u d i n g those institutes m a i n taining 'base' and 'active' collections 2.
IBPGR has p u b l i s h e d fourteen 'Directories of G e r m p l a s m Collections' c o v e r i n g food legumes, root a n d tuber crops, cereals, vegetables, industrial crops, fruits a n d tree nuts a n d forage crops. IBPGR does m a i n tain a database that contains a subset of the i n f o r m a t i o n in the 'Directories of G e r m p l a s m Collections' w h i c h i n c l u d e s c o n d i t i o n s of seed storage. It does not, h o w e v e r , p u b l i s h catalogues of collections, a l t h o u g h it has s u p p o r t e d their p u b l i c a t i o n b y other institutes. IBPGR has, for s o m e special exercises, c o m p i l e d passport, and, in s o m e cases, c h a r a c t e r i z a t i o n i n f o r m a t i o n for s o m e crops. This t y p e of exercise is n o w a vital part of the crop n e t w o r k s a n d is not p e r f o r m e d at IBPGR H e a d q u a r t e r s . IBPGR has s u p p o r t e d w o r k on Frankel and Brown's 3 and Brown's 4 'core c o l l e c t i o n ' concept, but has not p e r f o r m e d i n - h o u s e testing of the concept. (This c o n c e p t selects 10% of a g e r m p l a s m collection w i t h the highest level of variability, for detailed e v a l u a t i o n and distribution.)
References 1 Bower, D. J. (1989) Trends Biotechnol. 7, 111-116 2 Anon (1989) The Case for Crop Networks, Geneflow, June, pp. 6-7, IBPGR 3 Frankel, O. H. and Brown, A. H. D. (1984) in Crop Genetic Resources: Conservation and Evaluation (Holden, J. H. W. and Williams, J. T., eds), pp. 249-257, IBPGR 4 Brown, A. H. D. (1989) in The Use of Plant Genetic Resources (Brown, A. H. D., Frankel, O. H. and Williams, J. T., eds), pp. 136-156, Cambridge University Press D. H. V A N S L O T E N M. C. PERRY J. K O N O P K A
IBPGR, Via delle Sette Chiese 142, 00145 Rome, Italy.
Editor's note Unfortunately, the brevity necessitated by the review f o r m a t m e a n t that m a n y of the valuable services p r o v i d e d by the organizations listed in the original article c o u l d not be listed in detail.
53
Protein-based medical adhesives R o b e r t L. S t r a u s b e r g and Rebecca P. Link
There are m a n y naturally occurring adhesive proteins w h i c h have potential for application in medicine and dentistry. Cloning and expression of their genes enables the modes of action of these proteins to be better understood and increases their availability for practical applications. This article concentrates on the adhesive protein from the blue mussel Mytilus edulis but also describes medical adhesives based on fibrin isolated from h u m a n blood. For many years adhesive chemists have been intrigued by the task of developing adhesive materials for in vivo medical applications in areas such as ophthalmology, plastic surgery, otology, cardiology, and neurology and orthopedics. Any adhesive material targeted for medical applications should possess the following general characteristics: • biocompatibility (non-toxic, low immunogenicity); • adherence and curing in moist environments; • non-interference with the natural healing process; • appropriate adhesive and cohesive properties.
considered as a natural adhesive for reuniting severed nerves 3'4. The use of fibrin sealants has increased dramatically in recent years (Box 1). For many applications, this material may be the adhesive of choice. However, fibrin adhesives are unlikely to have universal appeal in medical applications because the strength of the material (both adhesive and cohesive) is not adequate for many procedures, and also because fibrin sealants are derived from human blood (Box 1), and therefore might potentially carry viral contaminants 1°'17'18. In the United States, fibrin adhesive is prepared on small scale from the patient's own blood 1°'17'18, therefore eliminating
Researchers have focused on the use of natural proteins with adhesive properties• In this article, we discuss the genetic engineering and processing of adhesive proteins naturally produced by the mussel Mytilus edulis. We will also discuss, as a reference point, fibrin sealant systems: the greater depth of experience with the fibrin systems will illustrate the requirements and potential for other medical adhesives•
potential problems from contamination. However, this is a time consuming and costly approach. Therefore, the human origin of materials in fibrin sealants, a strong positive factor in biocompatibility, might also limit its use. While it would be desirable to genetically engineer components of the fibrin adhesive system to reduce the potential for contamination, the complex nature of fibrin would make this extremely difficult to achieve. This creates opportunities for other medical adhesives•
Adhesive materials synthesized by marine organisms Marine organisms including mussels and barnacles produce remarkable moisture-resistant adhesives. Since these are applied and cured in moist environments, they are potentially useful for many medical and dental applications. The blue mussel Mytilus edulis synthesizes a specialized polyphenolic adhesive protein which plays a key role in attachment to surfaces. The protein is located in a specialized thread-like structure called the byssus which also contains several other proteins including collagen. When secreted and applied, the byssal adhesive is highly cross-
:6
Fibrin sealant systems Fibrin sealant was the first proteinbased adhesive system marketed for in vivo applications. Fibrin, a key component of the natural hemostatic process, was first employed in surgical procedures during World War 11'2. Interest in this material revived during World War II when it was
I
"It's Robert L. Strausberg and Rebecca P. Link are at Genex Corporation, 16020 Industrial Drive, Gaithersburg, MD 20877, USA. @ 1990, Elsevier Science Publishers Ltd (UK)
0167 - 9430/90/$2.00
an a m b i t i o u s of the r e w a r d s
project
if w e ' r e
all r i g h t ,
but think
successful!"
TIBTECH - FEBRUARY 1990 [Vol. 8]
54
--Box I
The biochemistry of fibrin sealants and their application Since the Second World War, researchers have gained great insight into the biochemistry of the fibrinolytic and homeostatic systems. This has lead to the practical development of fibrin sealants containing purified protein components 5'6. These fibrin adhesives, currently marketed in Europe, are sold under tradenames such as Tissucol ®, Tisseel ® and Fibrin-Kleber Human Immuno ®. Fibrin sealant systems are currently being tested in diverse medical applications including nerve 7 and vascular a repair, opththalmic surgeries 9, cardiovascular grafts 1°, cartilage/bone grafting 11 and skin graft adhesion 12. Fibrin adhesive is generally used as a two component system. Component 1 contains at least 70 mg fibrinogen, 2-7 mg fibronectin, 10 units of Factor XIII, and 35 Fg of plasminogen and aprotinin in a l m l solution. The component is prepared by adding a solution of aprotinin to a lyophilized powder of the human proteins. Component 2 is a thrombin solution, prepared by adding a CaCI2 solution to lyophilized bovine thrombin. The time for the fibrin adhesive to set is control led by the amount of thrombin used in the formulation. The fibrinogen solution is viscous and upon mixing with the thrombin solution quickly sets to a firm, white gel. When this reaction occurs at the application site the sealant adheres to the tissue 5. The major protein in the fibrin adhesive is fibrinogen. This protein, with a molecular mass of 340 kDa, consists of six polypeptide chains (three pairs of nonidentical chains designated ~, [3 and 7) linked through disulfide
linked and cannot readily be analysed biochemically. However, substantial progress has been made in characterizing the non-cross-linked adhesive protein which is produced and stored in the phenol gland 19 21. The 130 kDa protein is rich in proline/ hydroxyproline, serine, threonine, lysine, tyrosine/3,4-dihydroxyphenylalanine (Dopa) and alanine, a composition which suits the protein for interaction with biological surfaces (Table 1). The protein is extremely hydroxyl-rich with 60-70% of the amino acid residues containing hydroxyl groups. A high proportion of its proline residues are post-translationally hydroxylated to either 3- or 4-hydroxyproline 2°. In addition, more than half of its tyrosine residues are converted to Dopa. The decapeptide sequence Ala-Lys-Pro/HypSer-Tyr/Dopa-Hyp-Hyp-Thr-DopaLys was found by peptide sequence analysis to occur repeatedly 21. Recently, formulations of the natural mussel adhesive protein (MAP) have been tested in ophthalmic applications 22'23 and the preliminary indications are that the adhesive has potential both in the treatment of corneal perforations and in epikeratoplasty.
bridges. The proteolytic action of thrombin releases peptides from the amino termini of the o~ and [3 chains, thereby stimulating spontaneous polymerization of the fibrin monomers. At this point in the reaction, the fibrin monomers are not joined by strong covalent bonds. However, thrombin in the presence of Ca 2+ also catalyses the activation of the zymogen, Factor XIII, to the transaminase, Factor XIIla. The Factor XIIla catalyses formation of stable 7-glutamyl-e-lysine cross-links, adding to the strength and stability of the fibrin clot. This in vitro process mimics the natural reaction that results in the formation of a fibrin clot. Details of the fibrinogenfibrin system have been reviewed 13. Other components in the fibrin sealant are included to aid in the adhesion process. Fibronectin, a natural cellular adhesive, enhances the adhesion of fibrin to tissue, possibly through cross-links to the major vertebrate structural protein, collagen 14,15. Aprotinin and plasminogen are not essential for the formation of fibrin clot but are included in fibrin sealant formulations to control fibrin biodegradability. Aprotinin suppresses fibrin degradation. The natural protease for fibrin is plasmin, activated by tissue plasminogen activator TM. Inclusion of an inhibitor like aprotinin slows the degradation process to allow for natural healing. The fibrin sealant does not interfere with natural healing and is eventually degraded. Plasminogen, a component of the fibrin sealant, assists in the complete degradation of the adhesive.
Cloned DNA encoding the mussel adhesive protein In order to further investigate its amino acid sequence and to develop a microbial system for expression of the mussel adhesive protein, the Genex research group prepared cDNA libraries from Mytilus edulis phenol gland mRNA 24 and identified sequences encoding portions of the adhesive protein. Analysis of these clones revealed that the mussel adhesive protein carries a family of related decapeptide and hexapeptide sequences arranged as tandem repeats. In general, the decapeptide raTable I
Potential adhesive interactions of frequently occurring amino acids in M y t i l u s e d u l i s adhesive protein A m i n o acid
C h e m i c a l bond
Serine Threonine Lysine Tyrosine Hydroxyproline Dopa
Hydrogen Hydrogen Ionic Hydrogen Hydrogen Metal complex formation
repeat units have the formula A-LysB-Ser/Thr-Tyr-Pro-Pro-Thr/Ser-TyrLys, where A is usually Ala or Pro and B is usually Pro. However, many sequence variations are observed. The most highly conserved residues in the various decapeptide repeat units are tyrosine and lysine, and proline at position 6. The DNA sequence analysis 24 confirmed the repetitive nature of the adhesive protein and revealed many related decapeptides and hexapeptides not previously identified by peptide sequencing 21. Microbial production of mussel adhesive protein In order to produce sufficient quantities of adhesive protein for studying adhesive mechanisms and to develop materials for medical and dental applications, Genex scientists have developed a microbial system to produce the protein 25. Although microbial expression of the mussel gene might be expected to be similar in many respects to the expression of other higher eukaryotic genes, some particular considerations were made. Firstly, because the protein is encoded by repetitive DNA, it had to be established that the coding material would be stable in the microbial host.
TIBTECH - FEBRUARY 1990 [Vol. 8]
55
--Fig. 1 2Ft L
: ~ G A L 1
highly expressed E. coli or yeast genes. However, our results 25 suggest that microbial cells can express foreign repetitive sequences well.
UAS
'~
//F-o~1TIS
PH05 signal
Hydroxylation ~D adhesive cDNA encoding protein
Am R
H terminator
Yeast vector for expression of mussel adhesive protein gene. This yeastE. coli shuttle vector is designed to direct high-level expression of foreign genes 25 It carries the yeast LEU2d gene for selection in leu2 yeast hosts and a
replication origin from the natural yeast 2# plasmid. Expression of the adhesive protein is regulated by a hybrid promoter carrying the upstream activation site (UAS) from the GAL 1 gene and the transcription initiation site (TIS) from the alpha-factor (MF-~I) gene. Transcription is terminated by a yeast glyceraldehyde-3-phosphate dehydrogenase terminator. Translation is initiated by a synthetic sequence carrying preferred yeast codons and encoding the yeast PH05 signal.
A yeast expression vector for the adhesive protein gene (Fig. 1) has been used to produce derivatives of the natural mussel adhesive protein ranging in molecular mass from 20 to 100 kDa at about 2-5% of total cell protein. Somewhat surprisingly, the highly repetitive coding sequences are very stable in yeast, even when carried on high copy number plasmids. Furthermore, we anticipated that production of the adhesive protein in E. coli or yeast might be limited because the natural repetitive coding sequences include codons which are not normally preferred in
Secondly, three amino acids, proline, tyrosine and lysine, comprise about 70% of the amino acids in the protein; therefore, efficient microbial expression was likely to be challenging. Thirdly, the need for post-translational hydroxylation would need to be addressed. The expression system developed at Genex was based on yeast. In general, the most efficient expression modules include a very well regulated promoter, a highly expressed yeast coding sequence fused to the repetitive gene and a transcription terminator.
--Fig. 2 OH
OH
Tyrosinase '& 02
OH2 I NH--CH--CO--
Tyrosine
0
Catechol oxidase .~
.,~ CH 2
I NH--CH--CO--
Dopa
7, 02 CH2
I NH--CH--CO--
o-Quinone
Post-translational modifications of tyrosine residues in the Mytilus edulis adhesive protein. Both the hydroxylation of tyrosine to Dopa and oxidation of Dopa to quinone can be catalysed by tyrosinases derived from mushrooms and bacteria.
The adhesive protein produced in yeast contains neither Dope nor hydroxyproline residues. The importance of hydroxyproline in byssal thread formation and adhesion is unknown. However, it has been suggested that hydroxyproline residues provide additional hydroxylgroups without losing the 'structure breaking' characteristics of proline. An alternative explanation might be that hydroxyproline residues are involved in stabilizing interactions between protein molecules in the byssal thread. Hydroxyproline residues do stabilize the triple helix of collagen 26. Collagen is a prominent protein in the mussel byssal thread 27 and may interact closely with the mussel adhesive protein. Dopa residues are quite unusual in proteins; it has been suggested that these residues may be crucial for moisture-resistant adhesion to surfaces and for cross-linking to build cohesive strength. Our studies with the genetically engineered adhesive protein support this; they show that cross-linking and moisture-resistant adhesion requires the presence of the reactive, oxidized form of Dope, quinone. To achieve moisture-resistant adhesion, there is a need for tyrosine residues to be hydroxylated. Fortunately, the post-translational modification of tyrosine to Dope and Dope to quinone can be catalysed in vitro by mushroom and bacterial tyrosinases (Fig. 2). Using a bacterial tyrosinase we have developed methods for in vitro production of the Dope-adhesive protein. The protocol is a modification of the procedures using mushroom tyrosinase described by Marumo and Waite 28. Ascorbic acid is added to reduce any quinone produced back to Dopa. The process produces an adhesive protein in which >50% of the tyrosine residues are converted to Dope. The Dope-adhesive protein is stable in an acidic solution or as a lyophilized powder. The Dope-protein is the preadhesive form of the protein. The adhesive properties are generated when the Dope residues are oxidized to quinone.
56
TIBTECH - FEBRUARY 1990 [Vol. 8]
--Fig. 3 a
Adhesive and cohesive properties The adherence of the engineered mussel adhesive protein in an aqueous environment to various surfaces including polystyrene, glass, hydrogel and collagen has been tested. In each case, activation to the quinone form of the protein was required for good surface adhesion. Adhesive protein bound to polystyrene was resistant to removal by washing with 0.9% NaC1, 0.5% SDS or Triton X-100, 1% acetic acid, 0.1 N NaOH, at 37°C, and sonication in water for 30 minutes. The cohesive strength of the engineered adhesive protein depends upon intermolecular cross-linking which requires formation of quinone residues. The nature of the chemical bond involved in cross-linking has not yet been determined, although it has been suggested that quinone residues could bond to the c-amino group of lysine through a Michael addition reaction. Quinone formation (and cross-linking ) can be achieved by either chemical or enzymatic oxidation of the Dopa residues. The enzymatic process permits regulation of cross-linking by altering the tyrosinase concentration. Importantly, certain formulations of the adhesive protein form gels following cross-linking that are similar in appearance to collagen and fibrin gels. Preliminary evidence suggests that cross-linking with other proteins will occur when these are present at the time of activation. The adhesive protein appears to have affinity for both soluble and insoluble collagen. Cross-linking the engineered adhesive protein with natural extracellular matrix proteins might enhance in vivo adhesive performance. In an in vitro model system in which moist collagen sheets were used as the substrate, the performance of the engineered adhesive was compared with that of fibrin adhesive. It was concluded that the engineered adhesive protein consistently had significantly greater strength than fibrin adhesive. The performance of the adhesive in vivo has been examined using a w o u n d healing model system in animals. The initial in vivo studies demonstrated that useful adhesive properties can be observed in less than one minute, and that adhesive strength increased for several hours
More recently, direct biochemical analysis has revealed Dopa residues b in structural/adhesive proteins from several species. For example, the marine polychaete Phragmatopoma californica lives in protective tubes C ~ formed by a combination of natural debris and secretions from the worm: Jensen and Morse 3° suggest that the Dopa residues in this protein may act through quinone tanning to stabilize the complex and form adhesive bonds. The putative egg shell pree i:i:i:i:i:i:i:l cursor proteins from the vitelleria of the liver fluke Fasciola hepatica 31'32 provide further examples of Dopacontaining proteins. These proteins, which apparently contribute to the sturdiness of the egg shell, are similar g •::i:i:i:i:!i:i:i:!i:i:i:!i:i:i:i:!:i:i:!:iI:!:i:i:!:i:i:!:i:i:!:i:i:!:i:i:i:!:i:i:•i:i:!:i:i:i!:i:i:toi:i:i:!the •i:i:!:i:imussel :i:!i:• adhesive protein in that they carry Dopa residues within Genetic constructions for adrepetitive peptide sequences. hesive proteins: (a) cloned cDNA Genetic engineering technology encoding a portion of the M y t i l u s can provide great flexibility in the e d u l i s adhesive protein (the seproduction of proteins for adhesive quence encodes repeating decaapplications. We have assembled peptide/hexapeptide sequence); synthetic genes encoding adhesive (b-d) tandem repeats of cloned proteins carrying repeated peptides cDNA encoding increasingly higher molecular weight forms of designed to enhance adhesion to the adhesive protein; (e) hybrid particular surfaces (Fig. 3). It might sequence encoding a cell attachalso be possible to enhance the ability ment peptide linked to the enginof engineered adhesive proteins to eered adhesive protein; (f) hybrid interact with cells in vivo by creating protein carrying multiple cell fusion proteins which include cell attachment domains derived from attachment sequences from extrafibronectin or other cell matrix cellular matrix proteins such as proteins; (g) adhesive protein fibronectin, laminin and vitronectin. carrying repeated peptide sequences designed to enhance These hybrid coding sequences (e.g. adhesion to particular surfaces. Fig. 3) can be assembled from (The coding sequence is assemcombinations of cDNA and synthetic bled from synthetic DNA.) DNA. Through this technology and careful protein design, it is likely that a new generation of biocompatible adhesive materials could be develafter initial application. Wound heal- oped and, during the next decade, ing in the presence of the adhesive these materials could become key components in surgica] and inteappears normal. grated w o u n d management proOther adhesive proteins grams. Microbial synthesis might well be applied to other structural/adhesive Acknowledgements proteins with moisture-~'esistant The Genex research on engineered properties. While the presence of adhesive proteins has been supDopa residues in the mussel adhesive ported by Phase I and Phase II SBIR protein is unusual, it is certainly not grants from the National Institute of unique. Indeed, quinone tanning of Dental Research. We gratefully proteins to achieve moisture-resist- acknowledge the contributions of our ance and to retard biodegradability colleagues in the Genex bioadhesive has been suggested before: in 1950, research group. Brown 29 noted evidence of quinone tanning in various structural proteins References of invertebrates including the byssus 1 Grey, E. G. (1915) Surg. Gyneco]. of Mytilus edulis and the egg case of Obstet. 21,452-454 the liver fluke, Fasciola hepatica. 2 Harvey, S. C. (1916) Boston Med.
TIBTECH- FEBRUARY 1990 [Vol. 8]
Surg. J. 174,658-659 3 Young, J. Z. and Medawar, P. B. (1940) Lancet ii, 126-128 4 Tarlov, I. M., Denslow, C., Swarz, S. and Pineles, D. (1943) Arch. Surg. 47, 44-57 5 Redl, H., Schlag, G. and Dinges, H. P. (1985) in Biocompatibility of Tissue Analogs Vol. 1 (Williams, D. F., ed.), pp. 135-157, CRC Press 6 Ellis, D. A. F. and Pelausa, E. O. (1988) J. Otolaryngol. 17, 74-77 7 Cruz, N. I., Debs, N., and Fiol, R. E. (1986) Plast. Reconstr. Surg. 78, 369-373 8 Barton, B., Moore, E. E. and Pearce, W. H. (1986) J. Surg. Res. 40,510-513 9 Rostron, C. K., Brittain, G. P. H., Morton, D. B. and Rees, J. E. (1988) Arch. Ophthamol. 106, 1103-1106 10 Dresdale, A., Bowman, F. O., Jr, Malm, J. R. et al. (1985) Ann. Thorac. Surg. 40, 385-387 11 Gersdorff, M. C. H. and Robillard, T. A. J. (1985) Laryngoscope 95, 1278-1280 12 Vibe, P. and Pless, J. (1983) Scan. I. Plast. Reconstr. Surg. 17, 263-264 13 Doolittle, R. F. (1984) Annu. Rev.
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Biochem. 53, 195-229 14 Williams, E. C., Janmey, P. A., Johnson, R. B. and Mosher, D. F. (1983) J. Biol. Chem. 258, 5911-5914 15 Barry, E. L. R. and Mosher, D. F. (1988) J. Biol. Chem. 263, 10464-10469 16 Lijnen, H. R. and Collen, D. (1987) Ann. Biol. Clin. 45, 198-201 17 Epstein, G. H., Weisman, R. A., Zwillenberg, S. and Schreiber, A. D. (1986) Ann. Otol. Rhinol. Laryngol. 95, 4O-45 18 Siedentop, K. H., Harris, D. M., Ham, K. and Sanchez, B. (1986) Laryngoscope 96, 1062-1064 19 Waite, J. H. and Tanzer, M. L. (1981) Science 212, 1038-1040 20 Waite, J. H. (1983) J. Biol. Chem. 258, 2911-2915 21 Waite, J. H., Houseley, T. and Tanzer, M. L. (1985) Biochemistry 24, 5010-5015 22 Robin, J. B., Picciano, P., Kusleika, R. S., Salazar, J. and Benedict, C. (1988) Arch. Ophthalmol. 106, 973-977 23 Robin, J. B., Picciano, P. and Benedict, C. V. (1988) in The Cornea Trans-
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actions of the World Congress on the Cornea lll (Cavanagh, H. D., ed.), pp. 55-58, Raven Press Stransberg, R. L., Anderson, D. M., Filpula, D. et al. (1989) in Adhesives from Renewable Resources: ACS Symposium Series 385 (Hemingway, R. W. and Conner, A. H., eds), pp. 453-464, American Chemical Society Strausberg, R. L. and Strausberg, S. L. (1988) PCT Patent Application No. US87/02663 Kivirikko, K. I. and Myllyla, R. (1980) in The Enzymology of Posttranslational Modification of Proteins (Freedman, R. B. and Hawkins, H. C., eds), pp. 53-104, Academic Press Mascolo, J. M. and Waite, J. H. (1986) J. Exp. Zool. 240, 1-7 Marumo, K. and Waite, J. H. (1986) Biochim. Biophys. Acta 872, 98-103 Brown, C. H. (1950) Nature 165, 275 Jensen, R. A. and Morse, D. E. (1988) J. Comp. Physiol. B. 158,317-324 Waite, J. H. and Rice-Ficht, A. C. (1987) Biochemistry 26, 7819-7825 Waite, J. H. and Rice-Ficht, A. C. (1989) Biochemistry 28, 6104-6110
A d d i t i o n a l i n f o r m a t i o n m a y be o b t a i n e d f r o m IBPGR H e a d q u a r t e r s , Via delle Sette Chiese 142, 00145 R o m e , Italy.
Genetic resource corrections We w o u l d like to p r o v i d e clarification on the t y p e of p l a n t genetic r e s o u r c e s i n f o r m a t i o n that is m a i n t a i n e d b y the I n t e r n a t i o n a l Board for Plant Genetic Resources (IBPGR). Most of the i n f o r m a t i o n p r e s e n t e d a b o u t IBPGR b y D. Jane B o w e r in her article ' G e n e t i c resources w o r l d w i d e ' (Ref. 1) was not accurate due to a shift in IBPGR p r o g r a m m e s a n d a misu n d e r s t a n d i n g in t e r m i n o l o g y . IBPGR does c o o r d i n a t e an intern a t i o n a l s y s t e m of p l a n t genetic r e s o u r c e centres. Both n a t i o n a l a n d i n t e r n a t i o n a l institutes participate. IBPGR p r o m o t e s seed genetic resource conservation by providing financial a s s i s t a n c e a n d scientific expertise, but the m a n a g e m e n t of the collections is p e r f o r m e d b y each institute. Currently, h o w e v e r , IBPGR is not d e v e l o p i n g a n e t w o r k of active collections; rather it is facilitating the e s t a b l i s h m e n t of crop networks. T h e s e will e n c o m p a s s all interested parties i n v o l v e d w i t h an i n d i v i d u a l c r o p i n c l u d i n g those institutes m a i n taining 'base' and 'active' collections 2.
IBPGR has p u b l i s h e d fourteen 'Directories of G e r m p l a s m Collections' c o v e r i n g food legumes, root a n d tuber crops, cereals, vegetables, industrial crops, fruits a n d tree nuts a n d forage crops. IBPGR does m a i n tain a database that contains a subset of the i n f o r m a t i o n in the 'Directories of G e r m p l a s m Collections' w h i c h i n c l u d e s c o n d i t i o n s of seed storage. It does not, h o w e v e r , p u b l i s h catalogues of collections, a l t h o u g h it has s u p p o r t e d their p u b l i c a t i o n b y other institutes. IBPGR has, for s o m e special exercises, c o m p i l e d passport, and, in s o m e cases, c h a r a c t e r i z a t i o n i n f o r m a t i o n for s o m e crops. This t y p e of exercise is n o w a vital part of the crop n e t w o r k s a n d is not p e r f o r m e d at IBPGR H e a d q u a r t e r s . IBPGR has s u p p o r t e d w o r k on Frankel and Brown's 3 and Brown's 4 'core c o l l e c t i o n ' concept, but has not p e r f o r m e d i n - h o u s e testing of the concept. (This c o n c e p t selects 10% of a g e r m p l a s m collection w i t h the highest level of variability, for detailed e v a l u a t i o n and distribution.)
References 1 Bower, D. J. (1989) Trends Biotechnol. 7, 111-116 2 Anon (1989) The Case for Crop Networks, Geneflow, June, pp. 6-7, IBPGR 3 Frankel, O. H. and Brown, A. H. D. (1984) in Crop Genetic Resources: Conservation and Evaluation (Holden, J. H. W. and Williams, J. T., eds), pp. 249-257, IBPGR 4 Brown, A. H. D. (1989) in The Use of Plant Genetic Resources (Brown, A. H. D., Frankel, O. H. and Williams, J. T., eds), pp. 136-156, Cambridge University Press D. H. V A N S L O T E N M. C. PERRY J. K O N O P K A
IBPGR, Via delle Sette Chiese 142, 00145 Rome, Italy.
Editor's note Unfortunately, the brevity necessitated by the review f o r m a t m e a n t that m a n y of the valuable services p r o v i d e d by the organizations listed in the original article c o u l d not be listed in detail.
