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Someone has pointed out a scrambled sentence (a word processing problem) in
original article I distributed. Below is a corrected version, any confusion
about the makeup of the genetic constructions can be studied in the articles of
the reference list.If an explanation of the the gene additions from plants and
viruses including enhancers, leaders, introns and trascription terminators
would be helpful I will be glad to provide one.
Furthermore, I recently found that human lysozyme produced in rice was being
marketed. The technical complexities of the gene constructions described below
should not focus attention from the main problem.  Bio pharmaceuticals produced
in test release sites are being marketed. These products do not seem to have
faced much critical testing and even worse the secret test plots may have
polluted the food crops. If nothing is said the problem will surely grow.

April 12, 2004
Prof. Joe Cummins

?Genetically modified  biopharmaceutical products are being produced and
marketed in the United States of America?
        There has been a great deal of discussion about the testing of rice genetically
modified to produce  the human gene products lysozyme and lactoferrin in the
United States (1)  so far those tests are  in a stalemate. Nevertheless,
Sigma-Aldrich chemical company of the United States has been marketing  the
biopharmaceutical products trypsin, avidin, and  beta-glucuronidase (GUS)
processed from transgenic maize  for at least two years (2) while Prodigene
Corporation markets aprotinin (AproliZean)(3) as does  Sigma-Aldrich but from a
non-food or feed member of the tobacco family(4)  For example,. Trypsin is a
digestive enzyme  used extensively in research , to treat disease and in food
processing, the product TrypZean is marketed as an animal free product (to
avoid contamination with animal viruses or prions) it is produced jointly by
Sigma-Aldrich and Prodigene biotechnology company (Prodigene  is the company
that faced problems after contaminating  food crops with biopharmaceuticals in
the United States (5)). The production of genetically modified (GM) food crops
follows a strict  process in the United States, first controlled field tests
are undertaken for a number of seasons then  the proponent  applies for
deregulation of the  GM crop following reviews by the Animal Plant Health
Service (APHIS) of the United States Department of Agriculture (USDA), the Food
and Drug Administration (FDA) and the Environmental Protection Agency (EPA)
when the GM crop includes a plant incorporated bio-pesticide. Upon completion
of the process the GM crop is deemed to be de-regulated and can be grown
without monitoring. None of the GM crops producing the  products listed above
appear to have been deregulated. The production of those biopharmaceutical and
their marketing seems to have been accomplished without the benefit of final
regulatory approval.
        Production and marketing of the biopharmaceutical crops listed above was done
with apparently full cooperation of the FDA and USDA (the agriculture
department has  proprietary interest in some of the biopharmaceuticals).
Approval of the biopharmaceuticals and their production  appears to have been
done  using a tricky backdoor procedure based on a loophole in the regulation
of   field test releases.  According to Pew initiative on food and
biotechnology  ?current APHIS regulations do allow the commercialization of a
GE crop without a prior affirmative approval by the agency and without public
notice. Developers are not required to file a petition for non-regulated status
before they produce a plant commercially. It is possible for developers to grow
plants at a commercial scale under notification or field trial
permits, even if the plants might pose some identifiable environmental or human
health risk(6).? Crop production facilities are permitted as ?field tests?,
locations of such facilities  are designated confidential business information
and is not disclosed to  inhabitants even though  the genes and products of
such sites can easily contaminate  both crops, ground water and surface water.
There seems to be no direct way to find production facilities  to any but
producers and government regulators. The United States government  seems
committed to going ahead with a  procedure that by passes public input and
scrutiny and which if ,when finally revealed, threatens  the marketability of
US food exports. In contrast, the Canadian Food Inspection Service maintains
that ?plant products of test sites  cannot be marketed? (7) even though
numerous  plant biopharmaceutical products have been tested.
        Production of the commercial biopharmaceuticals was , for the most part,
achieved using maize. Maize is a crop of fundamental importance and should not
have been messed with in the way it has  to produce biopharmaceuticals. An
approach of that type might be excused on the basis that the products are
benign. However, the products are not benign. Exposure to such products will
result in characteristic impact on humans and animals. Each of the products
will be briefly reviewed below. But  first  the regulation of plant derived
biopharmaceuticals  was reviewed in 2000 (8). And in 2004 by the Pew Initiative
(6). Only the Pew report came to grips with  the practice of  marketing
virtually untested  products commercialized with out public input. A review of
the Prodigene products  was published in 2002 (9). As indicated earlier test
plot permits for  crops producing biopharmaceutical proteins  are usually
designated confidential business information so that the nature of the product
is hidden from the public , the exact location of test sites is not declared,
but APHIS does record  the crop and the state in which the modified crop is
tested. During 2003 and 2004  ProdiGene had test plots in Nebraska, Texas, Iowa
and   Missouri  (10), so residents of other  states can feel relieved about
their neighbors corn.
        Trypsin is an enzyme produced in the pancreas and it breaks down proteins in
digestion. The enzyme is used in a number of laboratory applications, it is
used in wound treatment and to treat diabetes. The enzyme is used in food
processing and often put into  infant formulations to aid in digestion. The
plant produced product is desirable because it is free of prions and animal
viruses.(11). The  method for modifying crop plants and preparation of the gene
for trypsin is described in US patent  6,087,558 (12). The gene for trypsin  is
isolated from bovine pancreas,  the gene is actually to produce  trypsinigen
which has six additional amino acids at the start of the enzyme (these amino
acids are cleaved to make active trypsin).The DNA inserted into the corn plans
comprises the ubiquitin promoter, including the first exon and intron; the
barley alpha amylase export signal sequence; a trypsinogen encoding sequence;
pinII terminator(potato protease inhibitor II terminator); 35S promoter and
terminator with the moPAT (maize optimized PAT) selectable marker. The PAT gene
is for glufosinate resistance the maize is herbicide tolerant but the gene is
mainly for selecting transformed cells.
        According to material data safety sheets provided  by trypsin manufacturers the
product is capable of causing allergy is a skin, eye and respiratory irritant
and may be a mutagen (13,14). Known allergens should not be produced in food
crops.
        Avidin  is a protein found in bird eggs, it functions to bind the vitamin
biotin which is required for  many insect pests. The pests are  inactivated by
the absence of the necessary vitamin. Maize transgenic for avidin production
is resistant to storage insect pests (16) Friends of the Earth  did an
excellent case study of avidin corn providing substantial evidence that the
protein caused dangerous biotin deficiency in humans and animals leading to
immune deficiency and growth retardation (17). Even marginal biotin deficiency
is teratogenic in mice (18)and implicated in human birth defects (19). Avidin
is used a great deal but mainly as a diagnostic tool. Commercial production
using transgenic maize  has been described in  a publication (20) and  in
patents (21,22).The genes used  in transgenic corn are similar to those
described above for trypsin. A chicken egg white avidin gene was modified in
genetic code to  make a maize optimum gene, The ubiquitin promoter  along with
first exon and intron were joined to  a barley signal sequence for localizing
avidin in the grain, after the avidin sequence  the potato protease inhibitor
II terminator sequence. A long with the avidin related a sequence with the
selectable Bar gene (glufosinate resistance )  driven by a double   CaMV
promoter, tobacco mosaic virus leader sequence and maize alcohol dehydrogenase
intron, the  transcription terminator was from the potato protease as above.
The avidin produced in maize has similar properties to avidin produced in egg
white but it is clearly a unique gene product that requires independent safety
assessment.
        Aprotinin is a protease inhibitor normally prepared from  bovine pancreas and
lung. Recombinant aprotinin  produced in plants is currently marketed as
indicated above. Bill Freese of friends of the earth provided  a very fine
review of the product and  the problem of allergy and pancreatic disease from
ingestion of  the biopharmaceutical (23). Some findings additional to those
covered by Bill Freese  are discussed as follows. Aprotinin is listed as a
reproductive hazard (24). There is serious danger to  those exposed to
aprotinin after having had a previous exposure. Fore example  a two year old
child suffered severe anaphylactic shock  after a  test dose of aprotinin (25).
 Fatal anaphylaxis  followed aprotinin exposure in a local application of
fibrin glue (26) or a similar application led to an immediate skin reaction
following re-exposure to fibrin sealent (27). Secret field testing  of  plant
based recombinant aprotinin could result in severe or fatal  anaphylaxis
either in a brief exposure in the maize field  of one previously treated during
surgery or exposure of one exposed to the maize field then treated during
surgery.
        Recombinant  aprotinin derived from the bovine gene  was produced in maize
(28,29). The genetic  construction was similar to those  described above for
maize based biopharmaceuticals. The ubiquitin promoter along with the ubiquitin
intron was linked to a signal sequence from barley amylase, the aprotinin
sequence was followed  by the transcription terminator for potato  protease II.
 A selectable  marker, bar (glufosinate tolerance) was added along with the
double CaMV promoter , tobacco mosaic virus omega leader and intron from maize
alcohol dehydrogensase along with the potato protease II  terminator . The
recombinant  aprotinin does not appear to have been extensively tested using
animals even though  its glycosylation pattern may differ from the animal
product and  effect its allergenicity and stability.
        The final commercial recombinant product  produced in maize  is
beta-Glucuronidiase (GUS). The gene is used in a wide array of experimental
situations  but does not appear to have therapeutic importance. It has been
observed that  formula milk for infants had a low content of GUS while mother?s
milk had elevated GUS. Elevated GUS has been implicated in bilirubinaemia
(jaundice) of breast fed infants (30) and breast fed infants of diabetic
mothers (31).  GUS is used extensively as a marker believed to have  liitle
effect on the phenotype of the test organism, however,  GUS was found to
enhance the feeding activity of the peach aphid (32) suggesting that the marker
may not be entirely without effect on the phenotype of organisms.
        Commercial production of  recombinant GUS  was  reported in 1998 (33). The
recombinant GUS gene was isolated from E. coli. The maize construction was
similar to those described previously  for the other recombinant proteins
including the promoters, introns etc. and selectable marker described above.
Codon alterations to accommodate  protein synthesis in plants, were  a
surprising omission  because bacterial genes do not normally perform well in
higher plants unless the gene codons are adjusted for the plant cell.
        In conclusion, the secretive production of truly dangerous pharmaceuticals in
food crops  is a truly disturbing development. Production of products such as
aprotinin may have fatal consequences to the unknowingly exposed The sale of
such products  without transparent public approval is a threat to  the
residents, not from the products themselves, but from the knowing exposure of
the public through the food crop and concealment of that information by
authorities. In a democratic society we should insure that elected local
officials  take care to insure that the secret production is not permitted in
the area. If the elected officials are uninterested or willing to connive with
the bureaucrats they should be replaced at election or by recall.
References
1.Cummins,J. ?Pharm crops near you? 2004 http://www.i-sis.org.uk/  pp1-5
2.Horn,M,Woodward,S. and Howard,J. ?Plant molecular farming:systems and
products? 2004 Plant Cell Reports ? 2004 in press DOI:
10.1007/s00299-004-0767-1
3.Prodigene ?Aprolizean recombinant aprotinin from maize?2004
http://www.prodigene.com/pdf/AproliZean(tm)%20Backgrounder.pdf
4.Yahoo Finance ?LSBC and Sigma-Aldrich announce an agreement to manufacture and
distribute plant-produced recombinant aprotinin? 2004
 http://biz.yahoo.com/bw/040308/85333_1.html
5.USDA NewsRelease ?USDA announces actions regarding plant protection act
violations involving prodigene,inc? 2002
http://www.usda.gov/news/releases/2002/12/0498.htm
6.Pew Initiative on food and biotechnology ?Issues in the regulation of
genetically engineered plants and animals? 2004 pp1-174
7.Perron,F. personal communication  CFIA 2004
8.Graham,S. ?Plant derived biologics meeting ? 2000 pp1-145
http://www.fda.gov/cber/minutes/plnt1040500.pdf
9.Hood,E. ?From green plants to industrial enzymes? 2002 Enzyme and Microbial
Technology? 30,279-83
10.APHIS ?Prodigene permit activity? 2004 APHIS-USDA 2004
http://www.aphis.usda.gov/cgi-bin/parse-company.pl  pp1-2
11.Prodigene ?TrypZean recombinant trypsin from maize? 2004 pp1-3
http://www.prodigene.com/pdf/TrypZean(tm)%20Backgrounder.pdf
12.Howard,J. and Hood,E. ?Commercial production of proteases in plants? United
States Patent 6,087,558 pp1-18
13. Safety data ?Safety (MSDS) data for trypsin from bovine pancreas? 2003 pp1-2
http;//physchem.ox.ac.uk/MSDS/TR/trypsin.html
14.Specialty Media ?MSDS EDTA,MSDS and trypsin MSDS?2004  pp1
http://www.specialtymedia.com/05Resources/MSDS/MSDS%20SM-2002-C.htm4/13/2004
1:39:46 PM
15. McGraw,L. ?Avidin an egg-citing insecticide protein in corn?  2000
Agricultural Research  http://www.ars.usda.gov/is/AR/archive/aug00/egg0800.htm
16. Kramer,K, Morgan,T, Throne,J, Dowell,F,  Bailey,M. and  Howard,J.
?Transgenic avidin maize is resistant to storage insect pests? 2000 Nature
Biotech 18,670-5
17. Freese,B. ?Biopharming:case study of avidin corn? 2002 Friends of the Earth
http://www.foe.org/biopharm/csavidin.pdf
18.Mock,D,Mock,N,Stewart,C,LaBorde,J. and Hansen,D. ?Marginal biotin deficiency
is teratogenic in ICR mice? 2003 J.Nutr.133,2519-25
19. Zemleni,J. and Mock,D. ?Marginal biotin is teratogenic  (in human)? 2000
Proc.Soc. Exp.Biol.Med.223,14-21
20. Hood,E,  Witcher,D,Maddock,S, Meyer,T, Baszczynski,C,  Bailey,M,  Flynn,P,
Register.J, Marshall,L, Bond,D,  Kulisek,E, Kusnad,A ,  Evangelista,R,
Nikolov,Z,
 Wooge,C, Mehigh,R,  Hernan,R, Kappel,W,  Ritland,D,  Ping ,C, Howard,L and
Howard,J. ?Commercial production of avidin from transgenic maize:
characterization of transformant, production, processing, extraction and
purification? 1997 Molecular Breeding 3,291-306
21. Bazynski,C,Hood,E,Maddox,S,Myer,T,Register,J,Witcher,D. and Howard,J.
?Commercial production of avidin in plants? 1998 United States patent 5,767,379
pp1-15
22.Albertson,M,Howard,J. and Maddox,S. ?Induction of male sterility in plants by
expression of high levels of avidin? 1999United States patent 5,962,769
23. Freese,B. ?Biopharming:case study of corn producing aprotinin? 2002 Friends
of the Earth  pp1-5  http://www.foe.org/biopharm/csaprotinin.pdf
24.Research Safety ?Appendix B:Reproductive Hazard? 2002 pp1-22
http;//www.northwestern.edu/research-safety/pdf
25. Ryckwaert,Y,Barthlet,Y,Bonnet-Boyer,M,Rochette,A,Capdevila,X. and d?Athis,F.
?Anaphylactic shock after a test dose of aprotinin in pediatric orthopedic
surgery? 1999 AnnFrAnesthReanim 18,904-8
26.Oswald,A,Joly,L,Gury,C,Disdet,M,Leduc,V. and Kanny,G. ?Fatal intraoperative
anaphylaxis related to aprotinin after local application of fibrin glue? 2003
Anasthesiology 99,762-3
27.Beierlein,W,Scheule,A,Antoniadis,G,Braun,C. and Schlosser,R. ?An immediate
allergic skin reaction to aprotinin after exposure to fibrin sealant? 2000
Transfusion 40,302-5
28. Zhong,G, Peterson,D, Delaney,D, Bailey,M, Witcher,D,  Register ,J,  Bond,D,
Li,C,  Marshall,L,  Kulisek,E, Ritland,D,  Meyer,T,  Hood,E. and  Howard,J.
?Commercial production of aprotinin in transgenic maize seeds? 1999 Molecular
Breeding 5,345-56
29. Baszczynski ,C, Czapla,T, Hood,E, Meyer,T, Peterson,D,Rao,G, Register,J,
Witcher,D. and Howard,J. ?Commercial production of aprotinin in plants? 1998
United States patent 5,824,870  pp1-15
30.Gourley,G. and Arend,R. ?beta-Glucuronidase and hyperbilirubinaemia in breast
fed and formula fed babies? 1986 Lancet 22, 644-6
31. Sirota,L, Ferrera,M,lerer,N. and Dulitzky,F. ?Betagucuronidase and
hyerbiirubinaemia in breast fed infants of diabetic mothers? 1992
Arch.Dis.Child 67,120-1
32. Cherqui A, Alla S, Saguez J, Doury G, Sangwan-Norreel B. and Giordanengo P.
?Probiotic effects of beta-glucuronidase on the peach-potato aphid Myzus
persicae (Aphididae)? 2003 J. Insect. Physiol. 49,1199-209
33.   Witcher,D, Hood,E, Peterson,D, Bailey,M, Bond,D, Kusnadi, A,
Evangelista,R, Nikolov,Z, Wooge,C, Mehigh,R, Kappel, W, Register,J. and  Howard
,J ? Commercial production of â-glucuronidase (GUS): a model system for the
production of proteins in plants? 1998 Molecular Breeding  4,301-12

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