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The chemical and biological properties of phomopsin
Toxicon, Supp1 .3, pp .149-152, 1983 . Printed in Great Britain .
Pergamon Presa Ltd .
THE CHEMICAL AND BIOLOGICAL PROPERTIES OF PHOI~PSIN John L . Prahn (1), Marjorie V . Jago (1), Claude C .J . Culvenor (1), John A . Edgar (1) and Alan J . Janes (2) (1) CSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No . 1, P .O . Parkvilla, Victoria, 3052, Australia ; (2) National NMR Centre, Australian National University, Canberra, A .C .T ., 2600, Australia INTRODUCTION Phomopsin (CULVENOR et al ., 1977) is a toxic metabolite of the fungus, Phomopais Zeptoatronriformis, that can infest lupins and cause the disease known as lupinosis in
sheep and cattle grazing the infected stubbles (GARDINER, 1967 ; MARASAS, 1974) . Lupinosis is an important problem in the sheep industry in IVestern Australia where lupins were initially used as a valuable crop in coastal areas . They are now increasingly grown in other areas for their high-protein seed, including the eastern states where the disease has also appeared . BIOLOGICAL PROPERTIES OF PHOMOPSIN In the field, characteristic features of lupinosis are anorexia, jaundice and or ochre-coloured, very fatty livers which are atrophic and cirrhotic in the more stages . Early signs may appear within 1 or 2 days of the introduction of a flock badly-affected lupin stubbles . Some animals may die within several days of onset signs, others may die later .
yellow chronic to of the
The field disease can be reproduced by the administration of phomopsin (JAGO et al ., 1982) isolated in crystalline form from toxic lupin roughage or seeds or, more conveniently, from liquid media in which the ftmgus has been cultured (LANIGAN et aZ ., 1979) . Sheep are extremely sensitive to the acute effects of the toxin, comparative studies having shown them to be about 100 times as sensitive as rats . (A lethal dose by subcutaneous injection to sheep is about 20ug/Kg body xeight) . The liver is the primary organ affected . In addition to the massive fat accumulation, a second characteristic of the phomopsin-poisoned liver is a colchicine-like arrest of mitosis (GARDINER and PETTERSON, 1972) . this metaphase arrest has been used as the basis for a sensitive bioassay using nursling rats (PETERSON, 1978) . The lower end-point of the assay measures a concentration of 300ng/ml, but levels as low as about 30ng/ml can be detected, though not reliably quantitated . One factor contributing to the hepatic lipid acc~ulation is a block in the secretion from the liver of triglycerides . Prolonged administration to rats of very low levels of phomopsin leads to a non-fatty but highly nodular liver consistent with the 'foxing-glove" type liver seen in chronic lupinosis in other species (MARASAS, 1974) . CHEMICAL PROPERTIES OF PHOMOPSIN In an earlier report (CULVENOR et aZ ., 1978) pho>oopsin was shown to be an acidic compound of approximate molecular formula, Cry Hwy Ns O1~ C1 . On the basis of mass and n . m . r . spectral evidence, the molecular formula has since been revised to C~ 6 Hy~ N6 Oii C1 (mol . wt . 770) . Phomopsin hydrolyses slowly (24 h) in dilute mineral acid at room temperature to oxalacetic acid and a product, phomopsinamine (HP 1), that apparently represents the re>aainder of the >solecule because it retains the spectral features and colour reactions of the parent compound . It was shown by paper electrophoresis to be an amine, and the fact that amino groups are liberated on hydrolysis indicates that the oxalacetic acid is linked in phomopsin as the di-amide . The formation of two other products under mild acid conditions may also be deaonstrated by paper electrophoresis :149
J . L . FRAHN et aZ .
150
A product intermediate between phomopsin and phomopsinamine forms rapidly in the presence of silver and mercuric salts . It is known that some amides are hydrolysed catalytically as their metal ion complexes (BUCKINGI~IAM et aZ ., 1974), and it is probable that one of the oxalacetamide bonds in phomopsin is broken in this way . The intermediate product is slowly converted to phomopsinamine when treated with dilute HCl . (ü) A methanol addition product or methyl ether, rapidly formed in methanolic HC1, is thought to be the enol ether of the oxalacetic entity . Electrophoretic evidence indicates that phomopsin loses an acid function in the process . In water, the product partly reverts to phomopsin, but changes also to phomopsinamine . In conc . HC1 at 50°C, phomopsinamine forms three other products, HP 2-4, whose maximum concentrations are reached in 1, 15 and 24 h, respectively . All contain phenolic and/or aromatic amine groupings . Simultaneously with the appearance of these products, ammonia (2 moles) and methylamine (0 .25 mole) are also liberated . The most likely sources of ammonia are carboxylic amide groups ; cyclic ureides such as barbituric acid are not hydrolysed under the above conditions . The less than molar yield of methylamine suggests the presence of a second closely related compound as an impurity in which -NHCH~ replaces -NHZ . Comparative electrophoretic mobilities of phomopsin, phomopsinamine and a number of reference compounds have been measured over a wide range of pH . The data suggest that phonopsin has an acid strength intermediate between those of carboxylic acids and simple phenols . Its anionic mobility in alkaline electrolytes is surprisingly high for a compound of relatively high molecular weight, and comparison with the reference compounds suggests that it has 4 acidic centres . the rise in anionic character in the range pH 7-10 resembles that of the aminophenols, but whereas these compoun ds are cationic below pH 7-8, phomopsin appears to have a very weakly basic group not ionised above pH 2 . By contrast, the hydrolysis product, phomopsinamine, has much lower anionic mobility but stronger cationic mobility, with the additional amine groups fully ionised at pH 4 .6 . Phomopsin and phomopsinamine couple with diazotised sulphanilic acid to give an orange-yellow dye, indicating a free ortho or pares position in a phenolic ring . Both compoinds undergo non-Malapradian oxidation by periodate to give a pink colour similar to that formed with p-methylaninophenol and p-aminophenol . Phomopsin also reacts with the ceric sulphate reagent of FARNSWORTH et aZ ., (1964) to give a pale yellow-orange coloua, changing to mushroom pink . Similar colours are formed by alkaloids containing an indole or dihydroindole nucleus .
Biosynthetic evidence . The addition of [ l4 C] intermediates to liquid cultures of P.Zeptoatromiformia has shown a specific incorporation of about 1~ of the activity from labelled phenylalanine, praline and isoleucine (A . L . PAYNE, private co~tnication) . Acetate, malate, propionate, sucrose and tryptophane were poorly incorporated .
The lack of incorporation of acetate eliminates the possibility of polyketides and rings of similar origin such as the A rings of flavanoids . Similarly, lack of propionate incorporation effectively eliminates suspected affinities between phomopsin and macro cyclic antibiotics of the ansamycin type (RINEHART and SHIELD, 1976) . Malate was considered a possible precursor of the oxalacetic grouping, but either it is not a precursor or it becomes too widely dispersed tries the tricarboxylic acid cycle for the incorporation to be made evident .
Mass apeetm .
Phomopsin has very low volatility and tndergoes rapid decomposition on a heated probe . Ions were observed only by fast scanning over short ranges about pre-set points . Under field desorption conditions, the highest mass ion groups had prominent peaks at m/z 771 and 785 (less intense) which are regarded as M + 1 ions of phomopsin and an i~urity or second component . In the presence of K+ ions, ions corresponding to M + K ions were observed at m/z 809 and 824, respectively . In the presence of Cs+ ions, an ion
Phomopein Characterization
151
m/z 903, corresponding to M + Cs for phomopsin, was observed . Other ions obtained from phomopsin were m/z 752 (752-758), 656 (655-660), 565-567, 293, 204-206, 172 (with s ~Cl isotope at 174) and 113 . Under rapid desorption electron impact conditions, ions m/z 770, 784 and 752 were observed . Thus the evidence strongly indicates 770 as the molecular weight of phomopsin, the sample also containing a second component of molecular weight 784 . A very strong m/z 113 fragment peak may be derived by elimination of the oxalacetic grouping as the cyclic imide or equivalent : M-113 = 657 . The hydrolysis product, phomopsinamine, is as unstable as phomopsin on the field desorption emitter . However, the rapid desorption electron impact technique gave an ion, m/z 657, with another peak at m/z 639 . Gas chromatography-mass spectrometry of the products of hydrolysis of phomopsin with conc . HC1 has given evidence of a C1-containing product of mol . wt . 172 . Accurate mass measurements made on the dimethyl ether of this compound by the method of BROPHY et aZ ., (1979) gave a molecular formula corresponding to C~ HS Os CT - .for the underivatised compound . The mass spectrum of the dimethyl ether shows ions m/z 200 (M + ), 185, 129, 114, 94 and 93 with s ~Cl isotope peaks at m/z 202, 187 and 131 . Possible formulations for this product are chlorohydroxybenzoic acid or chlorodihydroxybenzaldehyde . is C-N .M.R . apectm . Although the 1s C spectrum of phomopsin was previously considered to give support to the Css formula (CULVENOR et aZ ., 1978), further work has led to the redognition of 35 lines, one of which represents 2 carbon atoms, as determined by measurements with a long delay time . Thus the carbon number of phomopsin is 36 . Proton N . M.R, apectrwn . Phomopsin gives a well-resolved proton spectrum in ds-D[d50 as shown in Fig . 1 with annotated structural assignments .
OH ~NNCO-CFI~C-COHN
FIGURE 1 .
CH Z Cllà CO(t)
CH aW
PMR SPECTRUM OF PHOMOPSIN
Measurements of the N .M .R . and mass spectra together with other evidence outlined above have led to the postulation of the partial structure shown in Pig . 2 . This also contains a diagraaatic summary of some of the chemical reactions of phomopsin .
J. L . FRAHN et aZ .
15 2
MOL. FORMULA : C~H~Ns 011 CL (A~ox) MOL . WT. ~ 770 (E .I. and FD. M.S.) PARTIAL STRUCTURE SUGGESTED BY PRESENT EVIDENCE~2 1 -C-CH=~-CHaCH3-CH2 CHg-CFi2-~CHg-C=CCH3-~= CHOH 0-C-0 0~ O-C-N
DII..HCL ( 2 DAYS)
-~H-~H-N-IiC0
PHOMOPSINAMINE (H.PL) COOH CL
I I CH
u METHYL.ATION Ç-OH~ inHCL/CH3 OH CO
OXALACETIC ACID
CONC. HCL ( I-2 DAYS) H.P2--~H.P3, H.P4, NH3 , CH3NH2
( M. WT. 172)
FIGURE 2 .
PARTIAL STRUCTURE AND 501
REACTIONS OF PHOMOPSIN
REFERENCES BROPHY, J .J ., NELSON, D., GOLDSACK, R.J ., LIDGARD, R.O . and MELLEY, D.P . (1979) . Lctb . Pmet ., 28, 615-619 . BUCKINGHAM, D .A ., HARROWFIELD, J .MacB, and SARGESON, A.M . (1974) . J. Am . them . Soo . 96, 1726-1729. CULVENOR, C .C .J ., BECK, A.B ., CLARKE, M., LOCKRUM, P.A ., EDGAR, J .A ., FRAHN, J . L ., JAGO, M. V., LANIGAN, G .W ., PAYNE, A.L ., PETERSON, J .E ., PETEERSON, D .S ., SMITH, L .W . and WHITE, R.R . (1977) . Aunt . J. biol . Sci . 30, 269-277 . CULVENOR, C .C .J ., SMITH, L .W ., PRAHN, J .L . and LOCKRUM, P .A . (1978) . In : Effeeta of Poisottous PZanta on Irivestodc, pp . 565-573 (KEELER, R .F ., VAN KAMPEN, K.R . and Academic Press : New York . JAMES, L .F ., Eds .) . FARNSWORTH, N.R ., BLOMSTER, R .N ., DAMRA'IOSKI, D ., MEER, W.A . and CAMMARA1n, L .V . (1964) . LZoydia 27, 302-314 . GARDINER, M.R . (1967) . Adv . vet. Sai.. 11, 85-138 . GARDINER, M. R. and PETTERSON, D .S . (1972) . d . comp . Path . 82, 5-13 . JAGD, M. V., PETERSON, J .E ., PAYNE, A. L. and CAMPBELL, D . G. (1982) . Aunt . J . exp . BioZ . med . Soi . 60, 239-251 . LANIGAN, G .W .,PAYNE, A.L ., SMITH, L .W ., WOOD, P .McR . and PETEERSON, D .S . (1979) . AppZ . & emrinm. MiorobioZ . 37, 289-292 . MARASAS, W .F .O . (1974) . In : Myaotoxirta, pp . 111-127 (PURCHASE, I .F .H ., Ed .) . Elsevier : Amsterdam. PETERSON, J .E . (1978) . d. oomp . Path . 88, 191-203. RINEHART, K.L ., JR . and SHIELD, L .S . (1976) . Forteohr . Chem. org . Aatetoffe, 33, 231-307 .
Pergamon Presa Ltd .
THE CHEMICAL AND BIOLOGICAL PROPERTIES OF PHOI~PSIN John L . Prahn (1), Marjorie V . Jago (1), Claude C .J . Culvenor (1), John A . Edgar (1) and Alan J . Janes (2) (1) CSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No . 1, P .O . Parkvilla, Victoria, 3052, Australia ; (2) National NMR Centre, Australian National University, Canberra, A .C .T ., 2600, Australia INTRODUCTION Phomopsin (CULVENOR et al ., 1977) is a toxic metabolite of the fungus, Phomopais Zeptoatronriformis, that can infest lupins and cause the disease known as lupinosis in
sheep and cattle grazing the infected stubbles (GARDINER, 1967 ; MARASAS, 1974) . Lupinosis is an important problem in the sheep industry in IVestern Australia where lupins were initially used as a valuable crop in coastal areas . They are now increasingly grown in other areas for their high-protein seed, including the eastern states where the disease has also appeared . BIOLOGICAL PROPERTIES OF PHOMOPSIN In the field, characteristic features of lupinosis are anorexia, jaundice and or ochre-coloured, very fatty livers which are atrophic and cirrhotic in the more stages . Early signs may appear within 1 or 2 days of the introduction of a flock badly-affected lupin stubbles . Some animals may die within several days of onset signs, others may die later .
yellow chronic to of the
The field disease can be reproduced by the administration of phomopsin (JAGO et al ., 1982) isolated in crystalline form from toxic lupin roughage or seeds or, more conveniently, from liquid media in which the ftmgus has been cultured (LANIGAN et aZ ., 1979) . Sheep are extremely sensitive to the acute effects of the toxin, comparative studies having shown them to be about 100 times as sensitive as rats . (A lethal dose by subcutaneous injection to sheep is about 20ug/Kg body xeight) . The liver is the primary organ affected . In addition to the massive fat accumulation, a second characteristic of the phomopsin-poisoned liver is a colchicine-like arrest of mitosis (GARDINER and PETTERSON, 1972) . this metaphase arrest has been used as the basis for a sensitive bioassay using nursling rats (PETERSON, 1978) . The lower end-point of the assay measures a concentration of 300ng/ml, but levels as low as about 30ng/ml can be detected, though not reliably quantitated . One factor contributing to the hepatic lipid acc~ulation is a block in the secretion from the liver of triglycerides . Prolonged administration to rats of very low levels of phomopsin leads to a non-fatty but highly nodular liver consistent with the 'foxing-glove" type liver seen in chronic lupinosis in other species (MARASAS, 1974) . CHEMICAL PROPERTIES OF PHOMOPSIN In an earlier report (CULVENOR et aZ ., 1978) pho>oopsin was shown to be an acidic compound of approximate molecular formula, Cry Hwy Ns O1~ C1 . On the basis of mass and n . m . r . spectral evidence, the molecular formula has since been revised to C~ 6 Hy~ N6 Oii C1 (mol . wt . 770) . Phomopsin hydrolyses slowly (24 h) in dilute mineral acid at room temperature to oxalacetic acid and a product, phomopsinamine (HP 1), that apparently represents the re>aainder of the >solecule because it retains the spectral features and colour reactions of the parent compound . It was shown by paper electrophoresis to be an amine, and the fact that amino groups are liberated on hydrolysis indicates that the oxalacetic acid is linked in phomopsin as the di-amide . The formation of two other products under mild acid conditions may also be deaonstrated by paper electrophoresis :149
J . L . FRAHN et aZ .
150
A product intermediate between phomopsin and phomopsinamine forms rapidly in the presence of silver and mercuric salts . It is known that some amides are hydrolysed catalytically as their metal ion complexes (BUCKINGI~IAM et aZ ., 1974), and it is probable that one of the oxalacetamide bonds in phomopsin is broken in this way . The intermediate product is slowly converted to phomopsinamine when treated with dilute HCl . (ü) A methanol addition product or methyl ether, rapidly formed in methanolic HC1, is thought to be the enol ether of the oxalacetic entity . Electrophoretic evidence indicates that phomopsin loses an acid function in the process . In water, the product partly reverts to phomopsin, but changes also to phomopsinamine . In conc . HC1 at 50°C, phomopsinamine forms three other products, HP 2-4, whose maximum concentrations are reached in 1, 15 and 24 h, respectively . All contain phenolic and/or aromatic amine groupings . Simultaneously with the appearance of these products, ammonia (2 moles) and methylamine (0 .25 mole) are also liberated . The most likely sources of ammonia are carboxylic amide groups ; cyclic ureides such as barbituric acid are not hydrolysed under the above conditions . The less than molar yield of methylamine suggests the presence of a second closely related compound as an impurity in which -NHCH~ replaces -NHZ . Comparative electrophoretic mobilities of phomopsin, phomopsinamine and a number of reference compounds have been measured over a wide range of pH . The data suggest that phonopsin has an acid strength intermediate between those of carboxylic acids and simple phenols . Its anionic mobility in alkaline electrolytes is surprisingly high for a compound of relatively high molecular weight, and comparison with the reference compounds suggests that it has 4 acidic centres . the rise in anionic character in the range pH 7-10 resembles that of the aminophenols, but whereas these compoun ds are cationic below pH 7-8, phomopsin appears to have a very weakly basic group not ionised above pH 2 . By contrast, the hydrolysis product, phomopsinamine, has much lower anionic mobility but stronger cationic mobility, with the additional amine groups fully ionised at pH 4 .6 . Phomopsin and phomopsinamine couple with diazotised sulphanilic acid to give an orange-yellow dye, indicating a free ortho or pares position in a phenolic ring . Both compoinds undergo non-Malapradian oxidation by periodate to give a pink colour similar to that formed with p-methylaninophenol and p-aminophenol . Phomopsin also reacts with the ceric sulphate reagent of FARNSWORTH et aZ ., (1964) to give a pale yellow-orange coloua, changing to mushroom pink . Similar colours are formed by alkaloids containing an indole or dihydroindole nucleus .
Biosynthetic evidence . The addition of [ l4 C] intermediates to liquid cultures of P.Zeptoatromiformia has shown a specific incorporation of about 1~ of the activity from labelled phenylalanine, praline and isoleucine (A . L . PAYNE, private co~tnication) . Acetate, malate, propionate, sucrose and tryptophane were poorly incorporated .
The lack of incorporation of acetate eliminates the possibility of polyketides and rings of similar origin such as the A rings of flavanoids . Similarly, lack of propionate incorporation effectively eliminates suspected affinities between phomopsin and macro cyclic antibiotics of the ansamycin type (RINEHART and SHIELD, 1976) . Malate was considered a possible precursor of the oxalacetic grouping, but either it is not a precursor or it becomes too widely dispersed tries the tricarboxylic acid cycle for the incorporation to be made evident .
Mass apeetm .
Phomopsin has very low volatility and tndergoes rapid decomposition on a heated probe . Ions were observed only by fast scanning over short ranges about pre-set points . Under field desorption conditions, the highest mass ion groups had prominent peaks at m/z 771 and 785 (less intense) which are regarded as M + 1 ions of phomopsin and an i~urity or second component . In the presence of K+ ions, ions corresponding to M + K ions were observed at m/z 809 and 824, respectively . In the presence of Cs+ ions, an ion
Phomopein Characterization
151
m/z 903, corresponding to M + Cs for phomopsin, was observed . Other ions obtained from phomopsin were m/z 752 (752-758), 656 (655-660), 565-567, 293, 204-206, 172 (with s ~Cl isotope at 174) and 113 . Under rapid desorption electron impact conditions, ions m/z 770, 784 and 752 were observed . Thus the evidence strongly indicates 770 as the molecular weight of phomopsin, the sample also containing a second component of molecular weight 784 . A very strong m/z 113 fragment peak may be derived by elimination of the oxalacetic grouping as the cyclic imide or equivalent : M-113 = 657 . The hydrolysis product, phomopsinamine, is as unstable as phomopsin on the field desorption emitter . However, the rapid desorption electron impact technique gave an ion, m/z 657, with another peak at m/z 639 . Gas chromatography-mass spectrometry of the products of hydrolysis of phomopsin with conc . HC1 has given evidence of a C1-containing product of mol . wt . 172 . Accurate mass measurements made on the dimethyl ether of this compound by the method of BROPHY et aZ ., (1979) gave a molecular formula corresponding to C~ HS Os CT - .for the underivatised compound . The mass spectrum of the dimethyl ether shows ions m/z 200 (M + ), 185, 129, 114, 94 and 93 with s ~Cl isotope peaks at m/z 202, 187 and 131 . Possible formulations for this product are chlorohydroxybenzoic acid or chlorodihydroxybenzaldehyde . is C-N .M.R . apectm . Although the 1s C spectrum of phomopsin was previously considered to give support to the Css formula (CULVENOR et aZ ., 1978), further work has led to the redognition of 35 lines, one of which represents 2 carbon atoms, as determined by measurements with a long delay time . Thus the carbon number of phomopsin is 36 . Proton N . M.R, apectrwn . Phomopsin gives a well-resolved proton spectrum in ds-D[d50 as shown in Fig . 1 with annotated structural assignments .
OH ~NNCO-CFI~C-COHN
FIGURE 1 .
CH Z Cllà CO(t)
CH aW
PMR SPECTRUM OF PHOMOPSIN
Measurements of the N .M .R . and mass spectra together with other evidence outlined above have led to the postulation of the partial structure shown in Pig . 2 . This also contains a diagraaatic summary of some of the chemical reactions of phomopsin .
J. L . FRAHN et aZ .
15 2
MOL. FORMULA : C~H~Ns 011 CL (A~ox) MOL . WT. ~ 770 (E .I. and FD. M.S.) PARTIAL STRUCTURE SUGGESTED BY PRESENT EVIDENCE~2 1 -C-CH=~-CHaCH3-CH2 CHg-CFi2-~CHg-C=CCH3-~= CHOH 0-C-0 0~ O-C-N
DII..HCL ( 2 DAYS)
-~H-~H-N-IiC0
PHOMOPSINAMINE (H.PL) COOH CL
I I CH
u METHYL.ATION Ç-OH~ inHCL/CH3 OH CO
OXALACETIC ACID
CONC. HCL ( I-2 DAYS) H.P2--~H.P3, H.P4, NH3 , CH3NH2
( M. WT. 172)
FIGURE 2 .
PARTIAL STRUCTURE AND 501
REACTIONS OF PHOMOPSIN
REFERENCES BROPHY, J .J ., NELSON, D., GOLDSACK, R.J ., LIDGARD, R.O . and MELLEY, D.P . (1979) . Lctb . Pmet ., 28, 615-619 . BUCKINGHAM, D .A ., HARROWFIELD, J .MacB, and SARGESON, A.M . (1974) . J. Am . them . Soo . 96, 1726-1729. CULVENOR, C .C .J ., BECK, A.B ., CLARKE, M., LOCKRUM, P.A ., EDGAR, J .A ., FRAHN, J . L ., JAGO, M. V., LANIGAN, G .W ., PAYNE, A.L ., PETERSON, J .E ., PETEERSON, D .S ., SMITH, L .W . and WHITE, R.R . (1977) . Aunt . J. biol . Sci . 30, 269-277 . CULVENOR, C .C .J ., SMITH, L .W ., PRAHN, J .L . and LOCKRUM, P .A . (1978) . In : Effeeta of Poisottous PZanta on Irivestodc, pp . 565-573 (KEELER, R .F ., VAN KAMPEN, K.R . and Academic Press : New York . JAMES, L .F ., Eds .) . FARNSWORTH, N.R ., BLOMSTER, R .N ., DAMRA'IOSKI, D ., MEER, W.A . and CAMMARA1n, L .V . (1964) . LZoydia 27, 302-314 . GARDINER, M.R . (1967) . Adv . vet. Sai.. 11, 85-138 . GARDINER, M. R. and PETTERSON, D .S . (1972) . d . comp . Path . 82, 5-13 . JAGD, M. V., PETERSON, J .E ., PAYNE, A. L. and CAMPBELL, D . G. (1982) . Aunt . J . exp . BioZ . med . Soi . 60, 239-251 . LANIGAN, G .W .,PAYNE, A.L ., SMITH, L .W ., WOOD, P .McR . and PETEERSON, D .S . (1979) . AppZ . & emrinm. MiorobioZ . 37, 289-292 . MARASAS, W .F .O . (1974) . In : Myaotoxirta, pp . 111-127 (PURCHASE, I .F .H ., Ed .) . Elsevier : Amsterdam. PETERSON, J .E . (1978) . d. oomp . Path . 88, 191-203. RINEHART, K.L ., JR . and SHIELD, L .S . (1976) . Forteohr . Chem. org . Aatetoffe, 33, 231-307 .