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Uniwersytet Mikolaja Kopernika, Torun
1987
1992
Instytut Biologii Ogólnej i Molekularnej Lab.
Fizjologii Zwierzat Lab. Biofizyki
Fac de Sciences, Laboratoire de Neurophysiologie
Financial support exchange between
Universities Université dANGERS programme
SOCRATES European CommunitiesMinistere de
lEducation Nationale.....France Poste Past- 3
years co-tutelle grant- 3 years
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Form of collaboration
Students (cotutelle thesis)
Personal exchange
Teachers (teaching, research)
Study in the same subject - joint publications
1992 -2002 30 articles, 54 short com.
Participation in Conferences
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Electrophysiological Laboratories
Angers - Neurophysiol. Lab
Torun-Biophyscis Lab.
Cockroach isolated axon (oil gap) Cockroach
synapses (oil and mannitol gap) Cockroach
DUM cells (patch clamp) Oocytes of Xenopus
(microelectrode) Calcium imaging Vertebrate
muscles (oil gap)
Isolated axon (oil gap) DUM cells (in situ-
microelectrode) Cercal nerve (extra-
cellular electrodes) Invertebrate
muscles (set-up for excitation-contraction
coupling)
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Laboratory of Animal Physiology Insecticide
toxicity and temperature Modification of thermal
behaviour of vertebrates and invertebrates by
insecticides Thermal behaviour of animal after
openers and closers of sodium
channels Changes of animal metabolism under
pesticides -Long term experiments on different
species of insects and vertebrates
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Looking for new neurotoxins and potent
insecticides
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ISOLATION, PURIFICATION
TOXICITY TESTS
BINDING ESSAYES
ELECTROPHYSIOLOGICAL EXPS.
STRUCTURE-FUNCTION RELATIONSHIP STUDIES
PRODUCTION OF SYNTHETIC, RECOMBINANT
NEUROTOXINS AND THEIR MUTANTS
NEUROTOXIN APPLICATIONS - in neuroscience - in
agriculture - in drug teraphy
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INSECT SODIUM CHANNELS AS TARGETS FOR
ANTI - INSECT NEUROTOXINS
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CHARACTERIZATION
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SCORPION ANTI INSECT NEUROTOXINS
Alpha toxins
Excitatory toxins
AaHIT LqqIT1 LqhIT1 BjIT1 Bm33
Lqh?IT, Lqq?IT, BotIT1
BotIT2
Depressant toxins
LqhIT2, LqqIT2, BjIT2, BcTx1, BotIT4, BotIT5
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EXCITATORY TOXIN
DEPRESSANT TX
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RECOMBINANT TOXINS MUTANTS
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Effect of native AaHIT effect of recombinant
Bj-xtrIT
1982 Pelhate Zlotkin
1999 Froy et al.
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LqhaIT residues important for receptor site
recognition
Toxicity Unmodified 100 K8D
0.6 F17G 14 R18A 22 K62L 19 R64D 5 R64H 320
Zilberberg et al.., 1996
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Putative interaction surface of excitatory
toxins.
Bj-xtrIT
Froy et al.., 1999
AaHIT
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Mutants of depressant toxin LqhIT2
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Spider anti-insect toxins
?-ACTX-Hv1a (funnel-web spider Hadronyche
versuta) ? - palutoxin (Paracoelotes
luctuosus) Tx4(6-1) (armed spider Phoneutria
nigriventer)
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Advantages of toxin application by recombinant
baculovirus
1. - Toxins are chemically identical to the
native toxins
2. - Mimic the native toxin symptoms of
paralysis for example AaHIT spasmodic
contractions, progressive reduction of
mobility, a complete paralysis in a contracted,
shortened body shape
silkworm larvae Bombyx mori
Zlotkin et al.., 2001
3. - Modified toxin can be applied
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4. - Easy mode of application - easy access to
insect body -source of toxin on place -
toxin degradation and elimination less
important -accessible to CNS
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Result toxin potentiation Toxin applied by
baculovirus requires a much lower hemolymph
contents then injected native toxin to induce
similar insect paralysis - for AaHIT - 35 fold
potentiation
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Increased susceptibility of KDR insects to
anti-insect toxins
Paralytic potency of AaHIT to wild type (WT)
and KDR houseflies Susceptibility
ratio WT -1 KDR -9.2 sKDR -14 Zlotkin et al..,
1999
Heliothis virescens - Lee et al.., 1999
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DUM neuron background sodium channels modified by
anti-insect alpha toxin
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Activity of background sodium channels and
firing pattern in DUM neuron
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Motives for the study on animal neurotoxins
Application in agriculture
Curiosity
Immunotherapy
Application in medicine
Chemical scalpels to study biological processes
and targets
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BINDING SITES FOR INSECTICIDES IN SODIUM
CHANNEL
SITE 2 N-Alkylamides SITE 7 DDT analogues
PYRETHROIDS SITE 9 Dihydropyrazoles
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MUTATIONS IN INSECT SODIUM CHANNELS
ASSOCIATED WITH RESISTANCE TO PYRETHROIDS
kdr super-kdr L1029H-tobacco
budworm L918F L1014F-house fly L918F L993F-
German cockroach E434K C764R (alone-) V421M-tobac
co budworm
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MODE OF ACTION OF PYRETHROIDS
At 100 mV DDT 100mM - 9ms Tetramethr 20mM -
620ms Deltamethr 1mM - (? )
Song Narahashi, 1996
Pelhate et al.., 1990
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ALTERED PROPERTIES OF SODIUM CHANNELS
ASSOCIATED WITH GENETIC RESISTANCE TO
PYRETHROIDS
Acceleration of activation-deactivation in
sodium channels ballasted by pyrethroids
Drosophila Na channels with artificial kdr and
super-kdr mutations - Vais et al.., 2000
Decrease of neuron excitability
Tobacco budworm Heliothis virescens- Lee et al..,
1999
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SENSITIVITY OF INSECT MUTATED SODIUM
CHANNELS TO PYRETHROIDS
kdr and super-kdr mutations reduce the affinity
of pyrethroids for open channels
super-kdr mutations reduce the number of
deltamethrin binding sites per channel from two
to one.
Tobacco budworm Heliothis virescens- Lee et al..,
1999
Drosophila Na channels with artificial kdr and
super-kdr mutations - Vais et al.., 2000
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INSECT TRACHEAL SYSTEM