John C. Perez

Molecular basis of infrared detection by snakes

Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a ‘thermal image’ of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibres of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibres that innervate the pit organ. TRPA1 orthologues from pit-bearing snakes (vipers, pythons and boas) are the most heat-sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of transient receptor potential (TRP) channels as thermosensors in the vertebrate nervous system.

Resistance of warm-blooded animals to snake venoms.

Abstract Crotalus atrox venom resistance in 40 species of animal sera was studied by means of antihemorrhagic assay, ring precipitation test and serum protection test. The antihemorrhagic titers ranged from 0 to 256 with 16 animal sera having a titer greater than two. Five animal sera precipitated with C. atrox venom and the titers ranged from 0 to 64. Procyon lotor (racoon), Liomys irroratus (Mexican spiny pocket mouse) and two species of Peromyscus sp. (white footed deer mouse) were the only unimmunized animals with both a positive antihemorrhagic and ring precipitation titer. One of the resistant sera, Neotoma micropus (gray woodrat), was further studied to determine effects of five other snake venoms. Neotoma micropus serum neutralized hemorrhagic factors in all hemorrhagic venoms tested but would not neutralize the lethal factors of elapid venoms in the serum protection test. It appears that both reptiles and warm-blooded animals have antihemorrhagic factors in their sera.

Natural protease inhibitors to hemorrhagins in snake venoms and their potential use in medicine.

Snake venoms are complex mixtures of many toxins and enzymes which effectively immobilize prey without a struggle and assist in digestion. Certain animals have a remarkable resistance to envenomation of snakes. Naturally occurring factors that neutralize snake venoms have been found in the sera of most snakes and a few warm-blooded animals. These antihemorrhagic and antineurotoxic factors have been purified from snake and mammalian sera. The antihemorrhagins are not immunoglobulins since they have different physical and chemical characteristics. The natural immunity to hemorrhagins is the result of tissue inhibitors of metalloproteinases (TIMP) found in animal sera of resistant animals. Most animals have matrix metalloproteinases (MMP) and TIMP that are implicated in a wide variety of normal physiological processes and pathological conditions. MMP in animals have many biological functions in embryogenesis, morphogenesis and tissue remodeling. MMP activities are precisely regulated by endogenous TIMP. Disruption of the balance between MMP and TIMP causes various diseases such as arthritis, periodontal diseases, diabetes, ophthalmologic conditions, neoplasia, metabolic bone disease, atherosclerosis and orthopedic conditions. Resistant animals that have a high titer of TIMP would have a survival advantage when bitten by poisonous snakes. Snake venoms are abundant and stable sources of MMP which are medically important. The venom MMP which cause unregulated destruction of tissue have sequences which have some degree of homology with mammalian MMP which control normal biological processes. Resistant animals are important sources of TIMP which can be used to study metalloproteinase related diseases. For these reasons the MMP in snakes and TIMP in resistant animal are excellent candidates for developing new drug therapies.

Proteolytic, hemorrhagic and hemolytic activities of snake venoms

Proteolytic, hemorrhagic and hemolytic activities were tested on 47 different venoms from the Crotalidae, Viperidae, Elapidae, and Hydrophiidae families. Antihemorrhagic activity of crude opossum (Didelphis virginiana) and woodrat (Neotoma micropus) serum was tested against the venoms that presented hemorrhagic activity. All venoms showed proteolytic activity when non-specific substrates such as hide powder and collagen were used. Members of the Crotalidae family had the highest hide powder, chymotrypsin-like and hemorrhagic activity. However, members of the Elapidae family had the highest collagen activity. Hemolytic activity was present in 85% of the snake venoms tested. The crude opossum and woodrat serum neutralized the hemorrhagic activity of all the hemorrhagic venoms. Of particular interest is the poor correlation between the venom activities measured here and the phylogenetic position of the snake that possess them. This is particularly true at the genus and species level. Differences in activities were found among individuals of the same genus. The significance of these differences among venoms of closely related snakes is unknown. They do not seem to be adaptive, however little is known of the physiology and habits of most venomous snakes.

The purification and characterization of an antihemorrhagic factor in opossum (Didelphis virginiana) serum

Abstract Antihemorrhagic protein isolated from serum of the opossum (Didelphis virginiana) was homogeneous by polyacrylamide disc electrophoresis. The purified antihemorrhagic factor had an isoelectric pH of 4.1, a molecular weight of 68,000, migrated as the fastest band in disc electrophoresis, failed to form a precipitate with crude Crotalus atrox venom, and did not show proteolytic activity on gelatin. This evidence indicates that the antihemorrhagic factor in the serum of the opossum is albumin or closely associated with albumin.

Purification and characterization of a naturally occurring antihemorrhagic factor in the serum of the hispid cotton rat (Sigmodon hispidus)

Abstract The serum of the cotton rat contains a factor which neutralizes the hemorrhagic activity of Crotalus atrox venom. The antihemorrhagic factor was purified from S. hispidus by DEAE-Sephadex and flat-bed isoelectrofocusing. In the purified fraction there was one band as determined by disc electrophoresis and one symmetrical peak by ultracentrifugation. The purified antihemorrhagic factor had no gelatinase or caseinase activity and was stable at pH 3–10. The neutralization capacity of the purified preparation was approximately 20 times that of crude serum. The molecular weight was near 90,000 and the isoelectric point was 5·4. Neither the purified factor nor crude serum formed a precipitin line with C. atrox venom. The results suggest that the antihemorrhagic factor of S. hispidus is probably α-globulin, which has similar characteristics to the antihemorrhagic factors isolated in snake serum.

The resistance of three species of warm-blooded animals to western diamondback rattlesnake (Crotalus atrox) venom

Abstract The resistance of Sigmodon hispidus (hispid cotton rat), Neotoma micropus (gray woodrat), and Didelphis virginiana (opossum) to Crotalus atrox (Western diamondback rattlesnake) venom was studied by means of antihemorrhagic assay, hemorrhagic assay, mouse protection test and determination of the ld 50. The antihemorrhagic activity of serum and tissue extract was measured in the back of rabbits. In all three animals there was very little antihemorrhagic activity associated with tissue extract. There was antihemorrhagic activity associated with the three serum samples with the highest being D. virginiana. The hemorrhagic assay in the back of the three species of animals revealed that D. virginiana and N. micropus would neutralize the hemorrhagic factors in C. atrox venom at about the same rate. Sigmodon hispidus neutralized C. atrox venom but to a lesser degree. The ld 50 of the venom in S. hispidus was 172 mg/kg body weight which was 6·5 times less resistant than N. micropus but 21 times more resistant than white laboratory mice. The results clearly show that the three animals studied are resistant to rattlesnake venom.

The purification and characterization of an antihemorrhagic factor in woodrat (Neotoma micropus) serum

A protein antihemorrhagic factor was purified from the serum of the southern plains woodrat (Neotoma micropus) by gel filtration and ion exchange chromatography. The homogeneity of the purified factor was measured by disc electrophoresis in polyacrylamide gel. The factor had an isoelectric pH of 4.1, a molecular weight of 54,000, and migrated with alpha-globulins in disc electrophoresis. The factor failed to form a precipitate with crude western diamondback rattlesnake (Crotalus atrox) venom and did not show proteolytic activity with gelatin or hide powder, N. Micropus has an antihemorrhagic factor in the serum which appears similar to that found in hispid cotton rat (Sigmodon hispidus) and in opossum (Didelphis virginiana) sera.

Comparative study on hemorrhagic and proteolytic activities of snake venoms.

Abstract The hemorrhagic and proteolytic titers of nine snake venoms were measured. Suitable venom dilutions were selected to study the antihemorrhagic and antiproteolytic activities of three warm-blooded animal sera — gray woodrat (Neotoma micropus), hispidus cottonrat (Sigmodon hispidus) and Virginia opossum (Didelphis virginiana). It was demonstrated that all hemorrhagic venoms are proteolytic. The results show that the hemorrhagic activity of the venoms is readily neutralized by the sera; however, the proteolytic activity is not neutralized. We suggest that the mechanisms involved in neutralizing the two activities are not closely related.

Complementary DNA sequencing and identification of mRNAs from the venomous gland of Agkistrodon piscivorus leucostoma.

To advance our knowledge on the snake venom composition and transcripts expressed in venom gland at the molecular level, we constructed a cDNA library from the venom gland of Agkistrodon piscivorus leucostoma for the generation of expressed sequence tags (ESTs) database. From the randomly sequenced 2112 independent clones, we have obtained ESTs for 1309 (62%) cDNAs, which showed significant deduced amino acid sequence similarity (scores >80) to previously characterized proteins in National Center for Biotechnology Information (NCBI) database. Ribosomal proteins make up 47 clones (2%) and the remaining 756 (36%) cDNAs represent either unknown identity or show BLASTX sequence identity scores of <80 with known GenBank accessions. The most highly expressed gene encoding phospholipase A(2) (PLA(2)) accounting for 35% of A. p. leucostoma venom gland cDNAs was identified and further confirmed by crude venom applied to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and protein sequencing. A total of 180 representative genes were obtained from the sequence assemblies and deposited to EST database. Clones showing sequence identity to disintegrins, thrombin-like enzymes, hemorrhagic toxins, fibrinogen clotting inhibitors and plasminogen activators were also identified in our EST database. These data can be used to develop a research program that will help us identify genes encoding proteins that are of medical importance or proteins involved in the mechanisms of the toxin venom.