The origin of snakes and evolution of the venom apparatus.

The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and "dark" cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and "dark" cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and "dark" cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands. Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.

Morphology and ultrastructure of the venom glands of the northern pacific rattlesnake Crotalus viridis oreganus.

Among the myriad of enzymes present in animal venoms, nucleotidases and nucleases are poorly investigated. Herein, we studied such enzymes in 28 crude venoms of animals found in Brazil. Higher levels of ATPase, 5'-nucleotidase, ADPase, phosphodiesterase and DNase activities were observed in snake venoms belonging to Bothrops, Crotalus and Lachesis genera than to Micrurus genus. The venom of Bothrops brazili snake showed the highest nucleotidase and DNase activities, whereas that of Micrurus frontalis snake the highest alkaline phosphatase activity. On the other hand, the venoms of the snake Philodryas olfersii and the spider Loxosceles gaucho were devoid of most nucleotidase and DNase activities. Species that exhibited similar nucleotidase activities by colorimetric assays showed different banding pattern by zymography, suggesting the occurrence of structural differences among them. Hydrolysis of nucleotides showed that 1 mol of ATP is cleaved in 1 mol of pyrophosphate and 1 mol of orthophosphate, whereas 1 mol of ADP is cleaved exclusively in 2 mol of orthophosphates. Pyrophosphate is barely hydrolyzed by snake venoms. Phosphodiesterase activity was better correlated with 5'-nucleotidase, ADPase and ATPase activities than with DNase activity, evidencing that phosphodiesterases are not the main agent of DNA hydrolysis in animal venoms. The omnipresence of nucleotidase and DNase activities in viperid venoms implies a role for them within the repertoire of enzymes involved in immobilization and death of preys.

Nucleotidase and DNase activities in Brazilian snake venoms.

Secretion from Duvernoy's gland of the colubrid snakes Malpolon, Spalerosophis, and Thamnophis was obtained by pilocarpine stimulation and tested for lethality and selected enzymatic activities. Pools of secretion from Malpolon and Spalerosophis were fractionated by gel filtration, and several major active fractions were examined. The secretion from Malpolon had an LD50 of 6.5 micrograms/gm in mice; two lethal fractions with LD50's of 2.75 micrograms/gm and 4.5 micrograms/gm were isolated. One of these fractions appears to be a basic phospholipase A with a molecular weight of about 17,000. Spalerosophis had a secretion with an LD50 of 2.75 micrograms/gm in mice; one main lethal fraction with an LD50 of 2.5 micrograms/gm was isolated. Secretion from Thamnophis had an LD50 of 33.3 micrograms/gm; it was not fractionated owing to a paucity of material. At least two of the snake species examined have toxic secretions that may be important during feeding by killing or weakening and helping to subdue the struggling prey.

Lethal factors and enzymes in the secretion from Duvernoy's gland of three colubrid snakes.

Lysosomal diseases result from deficiency of one of the many enzymes involved in the normal, step-wise breakdown of macromolecules. Studies in vitro have shown that cells from enzyme-deficient patients can be corrected by an exogenous supply of the missing enzyme. This occurs by receptor-mediated endocytosis of normal enzyme added to tissue culture medium and also by direct transfer from normal leukocytes during cell-to-cell contact. Immunohistochemical analysis has revealed that these processes have similar pathways of intracellular transport of the acquired enzymes, which ultimately reach mature lysosomes in the recipient cells. Moreover, recent studies suggest that both mechanisms are important in the therapy of lysosomal storage diseases by bone marrow transplantation. Advances in gene technology are likely to improve the successful treatment of these disorders, by facilitating the large scale production of clinically effective proteins and also by enabling the stable and safe introduction of normal lysosomal genes into cells of affected patients.

Lysosomal storage diseases : mechanisms of enzyme replacement therapy

Duvernoy's gland (a type of venom gland) of the Japanese colubrid snake, Rhabdophis tigrinus, was examined by electron microscopy. The secretory units of the gland consist of the secretory and myoepithelial cells. The secretory cells are columnar in shape and have well-developed rough endoplasmic reticulum (RER), Golgi apparatus and mitochondria in the basal perinuclear cytoplasm. The secretory granules are homogeneous in structure and moderately dense. They are accumulated in the apical cytoplasm and are released by exocytosis into the lumen. The myoepithelial cells often enclose the secretory units, contain bundles of filaments in the cytoplasmic process, and are innervated by the free endings of the autonomic nerves through the basement membrane. In addition, many nerve terminals end in pericapillary spaces, suggesting a release of neurosecretions into the blood. The duct epithelium is composed of typical mucus secreting cells, whose cytoplasm contains secretory globules of lower electron density.

Fine Structure of Duvernoy's Gland of the Japanese Colubrid Snake, Rhabdophis tigrinus

Nerve fibers supplying the Duvernoy's gland, a venom-secreting oral gland, of the Japanese colubrid snake, Rhabdophis tigrinus, were examined by formaldehyde-induced fluorescence (FIF) and acetylcholinesterase (AChE) histochemistry, immunohistochemistry, and electron microscopy. The innervation by the FIF fibers was rather meager and was restricted to the area around the arteries localized in the interlobular connective tissue. The AChE-reactive fibers, in contrast, were abundantly supplied all over the gland, especially to the lobule consisting of secretory units. Peptide histidine isoleucine (PHI)(1-15)-like immunoreactive fibers were also detected in the gland and proved identical to the AChE-reactive fibers. The reactivities for both AChE and PHI (1-15) were particularly prominent around the blood capillaries distributed in the lobules. Under the electron microscope, nerve fibers were frequently seen to terminate near capillaries subjacent to the secretory unit. Those nerve terminals containing small clear vesicles and dense core granules were devoid of Schwann cell coverage on the side facing the blood capillary. These features suggest that the nerve terminals may possibly release at least a portion of their secretory contents into the blood as neurohormones.

Histochemical, immunohistochemical, and ultrastructural characteristics of nerves in the Duvernoy's gland of the Japanese colubrid snake, Rhabdophis tigrinus.

An electron microscope study of the Harderian gland of the snake, Rhabdophis tigrinus, revealed the occurrence of a myoid cell in the glandular body.The myoid cell, in oval profile, is located in the perivascular space and enveloped by a basement membrane. The plasma membrane of the cell is studded with a number of vesicular caveoli. The myoid cell cytoplasm is largely occupied by myofilaments which do not form discrete bundles of myofibrils. The striations comprise the A and I bands together with Z lines, but neither an M nor an H zone is detectable. Although the transverse tubule appears to be scarce, the sarcoplasmic reticulum is well developed. No triadic configuration is observed. The cytoplasm includes a few numbers of mitochondria, sarcoplasmic reticulum, fat droplets, dense membrane-bound granules and free ribosomes. The histological characteristics of the present myoid cell are compared to those of identical cells commonly existing in the thymus.

Occurrence of the myoid cell in the Harderian gland of the Japanese colubrid snake, Rhabdophis tigrinus.

The sea snake, E. ijimae, has no functional teeth other than maxillary fangs and pterygoid teeth in the jaws as has been observed in the present species by Voris (1966). Histological examination by light microscopy reveals that the dentary and palatine dental laminae exist close to the dentary and palatine bones respectively and the maxillary dental laminae is separated backward of the maxilla in the adult specimen. On the other hand, the fetus, nearly ready for birth, has tooth germs which are situated in superficial portion of the oral mucosa and also near the dentary bones. Because of these histological structures, the dentition of both specimens are apparently vestigial, and by taking the comparative anatomy and/or dietary habit into consideration, the present species of degenerated dentition may have originated from ancestral toothed species.

Tatsuyuki Ogawa

Papers

Fine Structure of Duvernoy's Gland of the Japanese Colubrid Snake, Rhabdophis tigrinus

Histochemical, immunohistochemical, and ultrastructural characteristics of nerves in the Duvernoy's gland of the Japanese colubrid snake, Rhabdophis tigrinus.

Occurrence of the myoid cell in the Harderian gland of the Japanese colubrid snake, Rhabdophis tigrinus.

Devolutive dentitions of the sea snake, Emydocephalus ijimae