**7. Molecular pharmacology and toxinology of** *D. quadriceps* **venom**

Recently, we have initiated a research project dedicated to investigate the composition, the pharmacological properties, and the transcripts from the venom gland components of *Dinoponera quadriceps.*

Using one-dimensional (SDS-PAGE) electrophoresis (1-DE) to resolve *Dinoponera quadriceps* venom proteins, only eight major large polypeptides (ranging from 15 to 100 kDa) were visualized by Comassie Brilliant Blue (CBB) Staining. The 1-DE and the insensitive method of staining with CBB was not adequate to separate small proteins below 15 kDa and peptides (Figure 3)

Collective efforts have been joined to annotate the gene composition of insects. The first complete sequenced genome of insect was from the fruit fly *Drosophila melanogaster*, in 2000, followed by a flurry of activities aimed at sequencing the genomes of several additional insect species. In the field of toxinology, the hymenopterans are receiving special attention due to

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Up to now, at least 10 ant species had their genomes analyzed and published. The ants whose genomes were sequenced include: the fire ant *Solenopsis invicta* found in South America, United States, China, Taiwan, Australia [49]; the Argentine ant *Linepithema humile*[50], the leaf-cutting ant *Acromyrmex echinator* [51] and *Atta cephalote* [52] found in South America; the red harvester *Pogonomyrmex barbatus* found in North and South America [53], the florida carpenter ant *Camponotus floriandus* from United States; and, the jumper ant *Harpegnatos saltator* from India, Sri Lanka and Southeast Asia [54]. Those ant genomes have provided hundreds of new

Apart of a detailed genome analysis, the construction of cDNA libraries from ants' venom glands is an important tool in order to analyze venom composition and discover new molecules that could have biological and pharmacological properties. But an important question arises: why hymenopteran venoms? As we pointed at the beginning of this chapter, there are several reports that hymenopteran venom could have biological properties useful for medical purpos‐ es. In this scope, from traditional and modern medicine reports, description can be found not only about clinical manifestation caused by hymenopterans venom, as allergic response, but al‐

Genomic and transcriptomic studies of hymenopteran cDNA libraries would provide useful information about their protein constituents. Some of these informations would include signal peptide sequences and the presence of post-translational modifications, which cannot be predicted by the studies of mature proteins. Ants genomic studies have shown a number of substances involved in the biology of these insects, such as: vittelogenins, gustatory and odorant receptors, molecules involved in immune response, as well as metabolic and structural

so the benefits of ant venom to treat disease like rheumatoid arthritis and pain [36].

**7. Molecular pharmacology and toxinology of** *D. quadriceps* **venom**

Recently, we have initiated a research project dedicated to investigate the composition, the pharmacological properties, and the transcripts from the venom gland components of

Using one-dimensional (SDS-PAGE) electrophoresis (1-DE) to resolve *Dinoponera quadriceps* venom proteins, only eight major large polypeptides (ranging from 15 to 100 kDa) were visualized by Comassie Brilliant Blue (CBB) Staining. The 1-DE and the insensitive method of staining with CBB was not adequate to separate small proteins below 15 kDa and peptides

their behavior and the ability to produce venom.

available nucleotide data.

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proteins like cytochrome P450.

*Dinoponera quadriceps.*

(Figure 3)

**Figure 3.** Electrophoretic profile of *Dinoponera quadriceps* total venom (DQv) in one-dimensional SDS-PAGE gel elec‐ trophoresis visualized with Comassie Brilliant Blue.

The peptide mass fingerprint (PMF), as well as other proteomic analysis is being conducted and a report will be published elsewhere.

Pharmacological studies have been realized with *Dinoponera quadriceps* venom, particularly, in a system of isolated perfused rat kidney. We now know that at concentrations of approxi‐ mately 10μg/mL increased urinary flow, glomerular filtration rate and decreased vascular resistance and sodium tubular transport, suggesting a natriuretic and diuretic effect. Further‐ more, in studies with renal tubule cells (MDCK - Madin-Darbin Canine Kidney) the same venom induced cell cytotoxicity, on MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte‐ trazolium bromide) at a dose and time dependent manner. Interestingly, greater cytotoxicity was observed in the shorter incubation periods, suggesting that the cell culture could recover after a given exposure time. Additional assays have been designed to evaluate the biological and pharmacological activity of purified component of this venom, as well as highlighting the mechanisms related to the observed effects.

**Figure 4.** Effect of *D. quadriceps* total venom (DQv) on Urinary flow (UF; A), sodium tubular transport percent(%pTNa; B) and renal vascular resistence (RVR; C). Ctrl=control. Results are expressed as means ± S.E.M., \*p<0.05 (ANOVA).

A part of proteomic and pharmacological studies, we prepared a *D. quadriceps* venom gland cDNA library to use an EST-strategy to identify the major transcripts expressed in the giant ant venom.we successfully constructed a full-length cDNA library of approximately 20 venom glands from *D. quadriceps,* using In-Fusion SMARTer kit (Clontech, USA). We obtained an

and stored at -80°C. A total of 432 individual ESTs were sequenced by the dideoxy chain termination (Sanger) method. Of these, 125 were undergone to a preliminary analysis through BLASTx. The Tabel 1 and Figure 8(A) shows an overview of the relative abundance of the protein groups.Most of the transcripts represent proteins involved in the whole metabolism as transferases, ATP synthase, dehydrogenases, ribosomal proteins, cytocrome c. Those sequences are being annotated for deposit in DNA and protein data bank. A note of caution is that, as in most trancriptome project, a significant number of transcripts showed no simi‐ larities with well-known sequences in data bank. These ESTs presents a typical structure of true ORFs (Open Reading Frame), that is start and stop codons, in addition a poly A tail. They were classified as (1) hypothetical proteins with unknown function and (2) cDNA precursors with no hits found. However, by comparing against DNA and protein data the hypothetical proteins showed high similarities with proteins from scorpions (*Opisthacanthus cayaporum)* and others ants, as *Harpegnatos saltator*, *Solenopsis invicta* and *Camponotus floriandus*. The Figure 8(B) represents the percentage of three classification of hits over the total clones analyzed, were probable toxins comprises a significant percentage of ESTs, representing about 34% of messages. Other 37% represents no-significant hits, which give us a number of perspectives

cfu/μg of DNA, our medium insert was 700bp and the library was amplified

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efficiency of 1x105

to analyze several novel proteins.

**Class Function % Clones** No hit Typical ORF with no hits 40.8 DnTx Mast cell degranulation 28.8 Hypothetical protein Unknown function 12.0 Antigen like Allergenic 9.6 Cytocrome c oxidase Metabolism 1.6 Cytocrome b Metabolism 1.6 Transferase Metabolism 2.4 Ionic channel blocker Toxin 1.6 Ribossomal protein Structural protein 1.6 Chymotripsin inhibitor Metabolism 0.8 Dehydrogenase Metabolism 0.8 ATP synthase Metabolism 0.8 Phospholipase A1 Enzyme/Toxin 0.8 Bacterial ESTs Symbionts (?) 4.0 Mitocondrial protein Metabiolism 0.8

**Table 1.** Classification of ESTs from *D. quadriceps* venom gland cDNA library on their putative functions.

**Figure 5.** Citotoxicity of *D. quadriceps* total venom on MDCK (Madin-Darbin Canine Kidney) cells culture on MTT assay. Results are expressed as means ± S.E.M., \*p<0,05 (ANOVA).

Recently we also demonstrated the neuroprotective activity of *D. quadriceps* venom in models of seizures induced by pentylenetetrazol (PTZ), when administered intraperitoneally. The effect was an increase in latency to first seizure and a tendency to increased latency of death, as well as reduction of lipid peroxidation in the prefrontal cortex of mice [55].

**Figure 6.** Effects of *D. quadriceps* venom (DQv) on latency of the first seizure in the models of seizure of pentylenete‐ trazol (PTZ) (A), pilocarpine (PILO) (B) and strychnine (STRC) (C). Results are expressed as means ± S.E.M., \*p<0.05 (AN‐ OVA).

**Figure 7.** Effects of *D. Quadriceps* total venom (DQv) on latency of death in models of seizure of pentylenetetrazol (PTZ) (A), pilocarpine (PILO) (B) and strychnine (STRC) (C). Results are expressed as means ± S.E.M (n=8), \*p<0.05.

A part of proteomic and pharmacological studies, we prepared a *D. quadriceps* venom gland cDNA library to use an EST-strategy to identify the major transcripts expressed in the giant ant venom.we successfully constructed a full-length cDNA library of approximately 20 venom glands from *D. quadriceps,* using In-Fusion SMARTer kit (Clontech, USA). We obtained an efficiency of 1x105 cfu/μg of DNA, our medium insert was 700bp and the library was amplified and stored at -80°C. A total of 432 individual ESTs were sequenced by the dideoxy chain termination (Sanger) method. Of these, 125 were undergone to a preliminary analysis through BLASTx. The Tabel 1 and Figure 8(A) shows an overview of the relative abundance of the protein groups.Most of the transcripts represent proteins involved in the whole metabolism as transferases, ATP synthase, dehydrogenases, ribosomal proteins, cytocrome c. Those sequences are being annotated for deposit in DNA and protein data bank. A note of caution is that, as in most trancriptome project, a significant number of transcripts showed no simi‐ larities with well-known sequences in data bank. These ESTs presents a typical structure of true ORFs (Open Reading Frame), that is start and stop codons, in addition a poly A tail. They were classified as (1) hypothetical proteins with unknown function and (2) cDNA precursors with no hits found. However, by comparing against DNA and protein data the hypothetical proteins showed high similarities with proteins from scorpions (*Opisthacanthus cayaporum)* and others ants, as *Harpegnatos saltator*, *Solenopsis invicta* and *Camponotus floriandus*. The Figure 8(B) represents the percentage of three classification of hits over the total clones analyzed, were probable toxins comprises a significant percentage of ESTs, representing about 34% of messages. Other 37% represents no-significant hits, which give us a number of perspectives to analyze several novel proteins.

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Results are expressed as means ± S.E.M., \*p<0,05 (ANOVA).

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OVA).

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as well as reduction of lipid peroxidation in the prefrontal cortex of mice [55].

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**Concentrations (g/mL)**

**Figure 5.** Citotoxicity of *D. quadriceps* total venom on MDCK (Madin-Darbin Canine Kidney) cells culture on MTT assay.

Recently we also demonstrated the neuroprotective activity of *D. quadriceps* venom in models of seizures induced by pentylenetetrazol (PTZ), when administered intraperitoneally. The effect was an increase in latency to first seizure and a tendency to increased latency of death,

**Figure 6.** Effects of *D. quadriceps* venom (DQv) on latency of the first seizure in the models of seizure of pentylenete‐ trazol (PTZ) (A), pilocarpine (PILO) (B) and strychnine (STRC) (C). Results are expressed as means ± S.E.M., \*p<0.05 (AN‐

**Figure 7.** Effects of *D. Quadriceps* total venom (DQv) on latency of death in models of seizure of pentylenetetrazol (PTZ) (A), pilocarpine (PILO) (B) and strychnine (STRC) (C). Results are expressed as means ± S.E.M (n=8), \*p<0.05.

\* \*

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**Table 1.** Classification of ESTs from *D. quadriceps* venom gland cDNA library on their putative functions.

**8. Conclusion**

**Acknowledgements**

**Author details**

Fortaleza, Brazil

**References**

Ceara, Fortaleza, Brazil

Toxicon 2006; 47(3):255-9.

A.F.C. Torres1\*, Y.P. Quinet2

CNPq/CAPES and FUNCAP for financial support.

, A. Havt3

2 Laboratory of Entomology, State University of Ceara, Fortaleza, Brazil

4 Marine Science Institute, Federal University of Ceara, Fortaleza, Brazil

\*Address all correspondence to: alba.fabiola@gmail.com

Taking into account the information presented in this chapter, a second question arises and should be answered in the near future: "Is there any hymenopteran venom component that could be used as a biotechnological tool?" The majority of works done to discovery new bio‐ technological tools from hymenopteran venoms were performed using proteomic science analysis, probably because ants apparatus venom is so hard to identify and dissect. Never‐ theless, the size of some poneromorph primitive ants may permit subdue these difficulties allowing us to construct a cDNA library and thus opening new perspectives to better under‐ stand the biology of ants as well as to analyze the properties of the venom in the search for

Thus, its clear that further work is necessary to understand ant venom, as well venoms from hy‐ menopteran, since several precursors comprises hypothetical and predicted toxins/polypepti‐ des with unknown function. Moreover, a deep functional analysis in the coming period will be

, G. Rádis-Baptista4

3 Biomedicine Institute, Department of Physiology and Pharmacology, Federal University of

[1] Ménez A, Stocklin R, Mebs D. 'Venomics' or: The venomous system genome project.

1 Departament of Clinical and Toxicological Analysis, Federal University of Ceara,

and A.M.C. Martins1

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made to comprehend the effects presented by total venom and peptides isolated from it.

new molecules with pharmacological and / or biotechnological potential.

**Figure 8.** Classification of ESTs from *D. quadriceps* venom gland cDNA library on their putative functions (A). Relative proportion of toxin-encoding, non-toxing encoding and no significant hit ESTs (B).

As a matter of example, the most abundant toxin was dinoponera toxin (DnTx). The dinopo‐ neratoxin whole sequence (accounting for 27% of the total clones analysed) was identified in this cDNA library. Deduced aminoacid sequences (DnTx01 and DnTx02), corresponding to two cDNA isoform precursos, from *D. quadricipes* transcriptome (this work) and three mature venom peptides (DnTx\_Da-3105, DnTx\_Da-3177 and TX01\_DINAS - GenBank accession numbers GI:294863162, GI:294863159 and GI:294863158, respectively) from *D. australis* [30] were aligned with ClustalW software using default parameters (http://www.ebi.ac.uk). DnTx01 and DnTx02 are represented with their respective signal peptides and pro-peptides, in which putative cleavage sites are shown in green and blue, respectively, according to SignalP software (http://www.cbs.dtu.dk/services/SignalP) and proteomic data. In the alignment A is clearly observed that DnTX01 shares high similarity with DnTx\_Da-3105 and DnTx\_Da-3177, whereas the mature DnTx02 and TX01\_DINAS are highly similar to each other (part B).

**Figure 9.** Alignment of dinoponeratoxin precursors and mature peptides from *D. quadricipes* and *D. australis* using ClustalW software (http://www.ebi.ac.uk).
