1. Identification of the role of the α1- and ß-adrenoceptors in the venom gland of the Brazilian snake Bothrops jararaca (Viperidae).
Bastos CMV1,3, Yamanouye N2 , Laia M5, Ho PL1,3,4 , Junqueira de Azevedo ILM1,3
1Centro de Biotecnologia and 2Laboratório de Farmacologia, Instituto Butantan, SP, Brasil ; 3Instituto de Biociências and Instituto de Química4, Universidade de São Paulo, SP, Brasil; 5Departamento de Engenharia Florestal, Faculdade de Ciências Agrárias, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG
Introduction
Methods
The venom gland of the Brazilian venomous snake Bothrops jararaca (Crotalinae, Viperidae) is an exocrine tissue related to the salivary gland. When the venom is released, the production of new venom is triggered by the activation of noradrenaline on both α1- and β-adrenoceptors. But the genes involved and the regulation of venom production cycle are poorly known. Our group has used the macroarray approach and bioinformatics tools to identify and characterize the differentially expressed genes of Bothrops jararaca venom glands during the venom production cycle and under specific treatments to inhibit the adrenocptors activity. The time series analysis showed that translation is the major function activated in venom gland at 4° day after the venom milking. Unespectely, the analysis of the differential gene expression showed that the venom gland treated with a stronger blocker of the venom production (reserpine) had a high level of toxins transcripts up-regulated. Real-time analysis confirmed these results and we hypothesized that the α1- and β-adrenoceptors could be involved, not in the transcriptional control of the toxins but in control of the venom secretion.
The cDNAs arrays were constructed with 4608 clones from male and female B. jararaca venom gland´s cDNA libraries, using the metodology that consist of bacterial clones grown in nylon membrane.
For time series analysis the venom gland was removed from snakes which venom was not manually extracted (0 days), and from snakes which venom was extracted 1, 2, 4 and 15 days before sacrifice. We used 3 snakes, all adult males, for each experimental condition.
A group of 3 male snakes were treated with reserpine, another group was treated with reserpine and with agonists of the adrenoceptors, and a control group receive no treatment. All snakes were milked and after 4 days killed for venom glands extraction.
The image obtained was scanned and analyzed using Bzscan 2 software. All statistical analysis were performed using R language and the package LIMMA from Bioconductor project. Clones with fold change > 2 (time series) and p-value >0,001 (treatments) were deemed diferentially expressed and selected for the next analysis.
Results
Significant gene expression pattern of the cellullar transcripts identified using the Short Time-Series Expression Miner (STEM) tool.
Functional annotation perfomed using the web tool DAVID (The Database for Annotation, Visualization and Integrated Discovery).
Biological Processes
number of genes
translational elongation
48 (16,5%)
RNA processing
20 (6,9%)
intracellular transport
19 (6,5%)
protein transport
15 (5,15%)
ribosome biogenesis
13 (4,5%)
mRNA metabolic process
10 (3,4%)
KEGG PATHWAY
Ribosome
44 (15,1%)
Ubiquitin mediated proteolysis
4 (1,3%)
Proteasome
3 (1,0%)
Molecular Function
structural constituent of ribosome
43 (14,8%)
structural molecule activity
49 (16,8%)
RNA binding
39 (13,4%)
transcription regulator activity
12 (4,1%)
cytoskeletal protein binding
8 (2,8%)
GTPase activity
ubiquitin-protein ligase activity
What these genes do?
Figure 1: the colorful lines represent the variation of expression of cellular transcripts from 0 to 15 days.
What happen when we “block” the venom production?
*A reaction of Real-Time PCR later...
MHF3 – metalloprotease HF3; VEGF – vascular endothelial growth factor; Boju III – Bojumet III;
4d – control untreated; 4dA – treated with reserpine +agonists; 4dR – treated with reserpine;
*Previous results of one experiment using a cDNA pool of 3 animals for each condition. Relative quantitation of toxins expression – ΔΔCT method using GAPDH as housekeeping.
We have more toxins...
Toxins are produced but not secreted?
*Another reaction of Real-Time PCR later...
Conclusion...
Our results provide the first evidence that the role of the α1 – and β – adrenoceptors in the snake venom glands may be in the control of the venom secretion rather than in the control of protein synthesis.
*Previous results of one experiment using a cDNA pool of 3 animals for each condition. Relative quantitation of toxins expression – ΔΔCT method using GAPDH as housekeeping.
Financial Support
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