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PubMedDate not supplied

Further observations on the pathological responses of rat skeletal muscle to toxins isolated from the venom of the Australian tiger snake, Notechis scutatus scutatus.

1. Some aspects of the response of mammalian skeletal muscle following the injection of purified toxins from the venom of the Australian tiger snake, Notechis scutatus scutatus, are described. 2. The toxins used were notexin, notechis II-5, notechis II-1 and a modified form of notexin (PBP-notexin). They were injected into the dorso-lateral aspect of one himd limb so that the soleus muscle would be exposed to the toxins. 3. Within 1 h after the injection of notexin, the soleus muscles were oedematous and by 3--6 h, polymorphonuclear leucocytes had entered the interstitial spaces. The invasion of necrotic muscle fibers was extensive by this time. Muscle spindles appeared relatively unaffected by the toxin. 4. The muscle regenerated via myoblasts at 2--3 days to myotubes at 3--5 days, immature muscle fibers at 7--14 days and fully differentiated muscle fibers by 21--28 days. Even after 6 months, however, the nuclei of many muscle fibres remained in a central position. 5. A second component of Australian tiger snake venom was also found to be myotoxic. It was slightly less potent than notexin, but caused qualitatively similar damage to that caused by notexin. It was identified as notechis II-5. A third fraction, notechis II-1, was found to be inactive. 6. Notexin could be neutralized by incubation with tiger snake antivenene; the simultaneous injection of antivenene with notexin did not afford complete protection against muscle damage.

Animals↗
PubMed2006

In vitro neurotoxic and myotoxic effects of the venom from the black whip snake (Demansia papuensis).

1. Black whip snakes belong to the family elapidae and are found throughout the northern coastal region of Australia. The black whip snake (Demansia papuensis) is considered to be potentially dangerous due to its size and phylogenetic distinctiveness. Previous liquid chromatography-mass spectrometry analysis of D. papuensis venom indicated a number of components within the molecular mass ranges compatible with neurotoxins. For the first time, this study examines the in vitro neurotoxic and myotoxic effects of the venom from D. papuensis. 2. Venom (10 microg/mL) caused significant inhibition of twitches elicited by stimulation (0.2 ms, 0.1 Hz, supramaximal V) of motor nerves in the chick biventer cervicis nerve-muscle preparation. This neurotoxic effect, which was postsynaptic in origin, was weak in comparison to that of most other Australian elapids. Prior addition (10 min) of polyvalent (PSAV) or tiger snake (TSAV) antivenom (5 units/mL) prevented venom-induced twitch inhibition. Addition of PSAV (5 units/mL) at t(50) failed to reverse the inhibitory effect but prevented further inhibition of nerve-mediated twitches. 3. The venom (20-50 microg/mL) is also myotoxic as indicated by a slowly developing contracture and inhibition of twitches elicited by direct stimulation (2 ms, 0.1 Hz, supramaximal V, in the presence of tubocurarine 10 micromol/L) of the chick biventer muscle. This activity was confirmed by histological examination of the muscle. 4. Fractionation and characterization of venom components is required to further investigate the reasons for the weak neurotoxic activity of D. papuensis venom.

Animals↗
PubMed1995

Responses of the aorta of the garter snake (Thamnophis sirtalis parietalis) to purines.

1. Isolated aortic rings from the garter snake (Thamnophis sirtalis parietalis) were investigated in order to identify and classify responses to adenosine and adenosine 5'-triphosphate (ATP) and their analogues as part of a comparative study of vertebrate purinoceptors. 2. Adenosine, D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R- and S-PIA) and 2-chloroadenosine (2-CA) all concentration-dependently relaxed aorta preconstricted with noradrenaline (NA). The order of potency was: NECA > R-PIA = 2-CA > adenosine > S-PIA. Individual pD2 values for the analogues were: NECA 7.12 +/- 0.13 (9), R-PIA 5.93 +/- 0.25 (7), 2-CA 5.64 +/- 0.40 (5), adenosine 5.04 +/- 0.10 (13) and S-PIA 4.26 +/- 0.10 (7). The order of potency has characteristics of both A1 and A2 receptors and cannot satisfactorily be classified according to the P1-(adenosine) purinoceptor subtypes established in mammalian preparations. 3. ATP, alpha, beta-methylene ATP (alpha, beta-MeATP), 2-methylthio ATP (2MeSATP), beta, gamma-methylene ATP (beta, gamma,-MeATP) and uridine 5'-triphosphate (UTP) all concentration-dependently constricted the isolated aorta. The order of potency was alpha, beta-MeATP = 2MeSATP > ATP > beta, gamma-MeATP > UTP. Only ATP, alpha, beta-MeATP and 2MeSATP consistently produced a maximum response; pD2 values were: ATP 3.98 +/- 0.07 (10), alpha, beta-MeATP 5.86 +/- 0.15 (12) and 2MeSATP 6.06 +/- 0.23 (9). In vessels preconstricted with NA neither ATP nor 2MeSATP caused relaxation in the presence or absence of the endothelium. 4. Suramin (0.1 mM) inhibited vasoconstriction to ATP, alpha,beta-MeATP, 2MeSATP and beta,upsilon-MeATP;however, since contractions to ATP and analogues did not reach a maximum response in the presence of this and other antagonists, pD2 values could not be calculated.5. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 micro M), a P2x-purinoceptor antagonist,antagonized constrictions to alpha, beta-MeATP only. Reactive blue 2 (RB2; 30 micro M), a P2Y-purinoceptor antagonist, inhibited vasoconstrictions to 2MeSATP only.6. Indomethacin (30 micro M) inhibited vasoconstriction in response to ATP and 2MeSATP, but not alpha,beta,-MeATP, suggesting that the presence of an unaltered phosphate chain on the ATP analogue was necessary to stimulate the production of a prostanoid.7. Repeated administration of alpha,beta-MeATP (3 microM) caused desensitization of the receptor responsible for the constriction due to alpha,beta-MeATP whereas the responses to ATP and 2MeSATP were unaltered.8. In summary, both P1-purinoceptors mediating vasodilatation and P2-purinoceptors mediating vasoconstriction are present on the garter snake aorta. However, in contrast to mammalian vessels, bothP2x and P2Y subtypes mediate vasoconstriction. There was no evidence for vasodilatation to ATP or analogues. Stimulation of the P2-purinoceptor by ATP and 2MeSATP caused the synthesis of a prostanoid. In addition, the possibility of a receptor activated by ATP, separate from P2X and P2Y subtypes is discussed since contractions to ATP proved to be insensitive to both PPADS and RB2. A comparison is made of purinoceptors in the garter snake aorta with those in other vertebrate vessels.

Adenosine↗
PubMed2006

Molecular evolution caught in action: gene duplication and evolution of molecular isoforms of prothrombin activators in Pseudonaja textilis (brown snake).

BACKGROUND: The evolution of structurally and functionally similar proteins with highly diverse physiological roles within a single organism is of great interest. Australian elapid snakes offer an excellent opportunity to study the molecular evolution of prothrombin activators. Venom from Pseudonaja textilis contains pseutarin C, a group C prothrombin activator. Its enzymatic subunit is structurally and functionally similar to mammalian factor (F) Xa, whereas its non-enzymatic subunit is similar to FVa. As vertebrates, the snakes also contain a system to activate prothrombin in their own blood during injury. These hemostatic factors are produced in the liver. RESULTS: Here we describe the presence of two molecular forms of FX expressed in the liver of P. textilis. Both isoforms have molecular signatures and domain architecture of FX. However, one isoform shows approximately 94% sequence identity with the snake FX from Tropidechis carinatus, whereas the other is much closer (90% identity) to the catalytic subunit of pseutarin C (PCCS). Real-time polymerase chain reaction reveals that the latter isoform is expressed approximately 56 000 times lower in the liver of P. textilis. However, the isoforms are not expressed in the venom gland. CONCLUSION: A detailed analysis of deletions and insertions along with the sequence indicates that the second isoform is an intermediate caught in the evolution of venom prothrombin activator from the blood coagulation FX. Thus, this isoform represents a 'molecular fossil' and reveals the likely evolutionary path of recruitment of FX in the venom gland.

Amino Acid Sequence↗
PubMed1998

Coronary artery bypass with "snake" grafts: fifteen-year follow-up.

BACKGROUND: Since 1970 we have used the "snake" graft in more than 8000 cases of coronary artery bypass grafting (CABG). We followed, for over 15 years, 214 patients who underwent CABG in 1981 with only a "snake" graft by one surgeon (William H. Sewell, M.D.). METHODS: Eighty-four percent (180/214) were male and 16% (34/214) were female with an average age of 58 and 66 years, respectively. The average left ventricular ejection fraction (LVEF) was > 55% in 63% (135/214) of the patients and < 55% in 37% (79/214); 15% (33/214) had diabetes and 77% (164/214) were smokers. The mean preoperative New York Heart Association (NYHA) functional class was 3.1+/-0.6 (range I-IV). Diagnostic arteriography demonstrated two vessel disease in 5% (9/214), three vessel disease in 89% (191/214), and left main disease in 6% (14/214). The average number of distal grafts per patient was 3.4. Coronary arteriography was performed 8-weeks postoperatively. Annual follow-up with a questionnaire determined incidence of redo procedures and survival. RESULTS: The perioperative 30-day mortality was 1% (2/214). At 8 weeks there was a graft patency rate end-to-side of 85%, side-to-side 97%, and the proximal segment of 95%. Four percent (9/214) had redo surgery while 6% (13/214) underwent angioplasty during the 15 years. Sixty-seven percent (144/214) were alive at 15 years with a significantly improved mean NYHA functional class of 1.2+/-0.9 (p < 0.001). Twenty-four percent of those 166 live patients (35/144) had an average LVEF of 50% by echocardiography. There were 34 (16%) noncardiac deaths, 32 (15%) cardiac deaths, and 2 (1%) unknown causes. CONCLUSIONS: The results of this study suggest that survival using the "snake" graft conduit is competitive with that observed using the internal mammary artery.

Aged↗
PubMed2005

Liver segmentation for CT images using GVF snake.

Accurate liver segmentation on computed tomography (CT) images is a challenging task especially at sites where surrounding tissues (e.g., stomach, kidney) have densities similar to that of the liver and lesions reside at the liver edges. We have developed a method for semiautomatic delineation of the liver contours on contrast-enhanced CT images. The method utilizes a snake algorithm with a gradient vector flow (GVF) field as its external force. To improve the performance of the GVF snake in the segmentation of the liver contour, an edge map was obtained with a Canny edge detector, followed by modifications using a liver template and a concavity removal algorithm. With the modified edge map, for which unwanted edges inside the liver were eliminated, the GVF field was computed and an initial liver contour was formed. The snake algorithm was then applied to obtain the actual liver contour. This algorithm was extended to segment the liver volume in a slice-by-slice fashion, where the result of the preceding slice constrained the segmentation of the adjacent slice. 551 two-dimensional liver images from 20 volumetric images with colorectal metastases spreading throughout the livers were delineated using this method, and also manually by a radiologist for evaluation. The difference ratio, which is defined as the percentage ratio of mismatching volume between the computer and the radiologist's results, ranged from 2.9% to 7.6% with a median value of 5.3%.

Algorithms↗
PubMed2006

Mast cells can enhance resistance to snake and honeybee venoms.

Snake or honeybee envenomation can cause substantial morbidity and mortality, and it has been proposed that the activation of mast cells by snake or insect venoms can contribute to these effects. We show, in contrast, that mast cells can significantly reduce snake-venom-induced pathology in mice, at least in part by releasing carboxypeptidase A and possibly other proteases, which can degrade venom components. Mast cells also significantly reduced the morbidity and mortality induced by honeybee venom. These findings identify a new biological function for mast cells in enhancing resistance to the morbidity and mortality induced by animal venoms.

Animals↗
PubMed1971

Chemical-cue preferences of newborn snakes: influence of prenatal maternal experience.

Newborn garter snakes (Thamnophis sirtalis) responded similarly to worm and fish surface extracts regardless of whether the mothers were fed exclusively on fish or worms during the gestation period. Worm extract was the more effective. This result is in contrast with the easy modifiability of chemical-cue preferences in young snakes after birth. The initial stimulus control of the attack response in newborn snakes thus seems relatively unmodifiable.

Animals↗
PubMed1999

Molecular analysis of the 18S rRNA gene of Cryptosporidium serpentis in a wild-caught corn snake (Elaphe guttata guttata) and a five-species restriction fragment length polymorphism- based assay that can additionally discern C. parvum from C. wrairi.

An adult wild-caught corn snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-microm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidium spp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on snake Cryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no. AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis (Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI and SspI, complements a previously reported assay by additionally distinguishing the bovine strain of Cryptosporidium from Cryptosporidium wrairi.

Animals↗
PubMed2006

Endoscopic examination of snakes by access through an air sac.

Sixteen boa constrictors (Boa constrictor), three royal pythons (Python regius) and 15 Burmese pythons (Python molurus bivittatus) were examined endoscopically by access through the air sac. The snakes were immobilised in a ventral position using a half-open anaesthetic system with assisted ventilation and a mixture of isoflurane and oxygen. The rigid endoscope was introduced percutaneously and the internal structure of the lungs and the air sac, and the shape, size and external surface of the liver were visible in the cranial direction. In the smaller snakes the bifurcation and caudal part of the trachea could be viewed, provided the endoscope was positioned in a retrograde orientation. The caudal orientation of the endoscope made it possible to view the gall bladder and the size, shape and surface of the spleen. In some cases, the pancreas and the surface of the stomach and colon could be monitored. Endoscopy through the air sac also made it possible to check the major veins in the coelom. The snakes were monitored for at least 30 days after the intervention and no changes in their respiratory function or general health were observed.

Air Sacs↗
PubMed1997

Intraerythrocytic inclusions associated with iridoviral infection in a fer de lance (Bothrops moojeni) snake.

Intraerythrocytic inclusions associated with infection by an iridovirus were observed in a fer de lance (Bothrops moojeni) snake that was being evaluated for the presence of renal carcinoma. The erythrocytes contained two types of inclusions, one viral and one crystalline, usually concomitantly. The snake was markedly anemic and exhibited a marked regenerative response. Ultrastructural analysis identified the virus to be an iridovirus consistent with snake erythrocyte virus and the crystalline structures to be of a different nature than hemoglobin.

Animals↗
PubMedDate not supplied

The association of conjunctival snake-like chromatin with keratoconjunctivitis SICCA.

PURPOSE: This work describes the snake-like chromatin cells in the conjunctiva of patients with keratoconjunctivitis sicca (KCS), assessing the relationship between tear function parameters and the severity of the disease, and searching for any differential pattern that could lead to early and diagnosis help clarify the etiology of KCS. METHODS: The conjunctiva of 366 eyes from 187 patients with KCS, and 30 eyes from 15 controls, were classified according to the results of the Schirmer I test, break-up time, rose Bengal staining, osmolarity, ferning tests and electrophoresis; they were then studied by impression cytology and electron microscopy. RESULTS: Snake-like chromatin cells, other nuclear changes, and a decreased number of goblet cells were found by impression cytology in different areas of the KCS conjunctiva. The presence of snake-like chromatin was significantly correlated with the severity of the disease. No nuclear changes were found in the control group. CONCLUSIONS: We assume these findings can be attributed to a natural regeneration of conjunctival cells and to resistance to a pathological reaction to KCS.

Adult↗
PubMed2006

North American coral snake antivenin for the neutralization of non-native elapid venoms in a murine model.

OBJECTIVES: North American coral snake antivenin (CSAV; Wyeth Antivenin [Micrurus fulvius], equine origin) is approved for the treatment of coral snake envenomations in the United States. The coral snake is the only elapid that is native to North America, but envenomations from non-native elapids are occurring more commonly in this country. This study was designed to evaluate the efficacy of CSAV in the neutralization of two exotic elapid envenomations: Naja naja (Indian cobra) and Dendroaspis polylepsis (black mamba). METHODS: A randomized, blinded, placebo-controlled murine model of intraperitoneal venom injection was employed. Venom potency was determined in preliminary dosing studies. Study animals then were divided into five groups: 1) N. naja venom + CSAV, 2) N. naja venom + 0.9% normal saline (NS), 3) D. polylepsis venom + CSAV, 4) D. polylepsis venom + NS, and 5) CSAV + NS. The venom dose was chosen to be twice the estimated LD50. The amount of CSAV injected was ten times the amount necessary for neutralization of a 2 x LD50 dose of M. f. fulvius venom in a murine model. Statistical analysis included Fisher's exact and log-rank testing to compare survival rates and times. RESULTS: Preliminary studies estimated the venom LD50 to be 2.58 mg/kg and 0.45 mg/kg, respectively, for the N. naja and D. polylepsis. A significant difference was shown in comparison of survival times between CSAV-venom groups and normal saline-venom groups despite all animals in both treatment and control arms dying. Animals receiving CSAV and N. naja venom survived (mean +/- SD) 24.4 +/- 3.0 minutes, versus 17.8 +/- 1.3 minutes in the control group (p < 0.001), whereas those receiving CSAV and D. polylepsis venom survived 203.8 +/- 37.0 minutes versus 130.0 +/- 42.6 minutes in the control group (p < 0.001). All animals in the CSAV + NS group survived to the conclusion of the study. CONCLUSIONS: When premixed with venom, CSAV increased survival time in a murine model of intraperitoneal N. naja and D. polylepsis venom injection. The clinical implications of this are unclear, given unchanged mortality rates.

Animals↗
PubMed2003

Sweeping and striking: a kinematic study of the trunk during prey capture in three thamnophiine snakes.

The trunk plays an obvious and important role in the prey capture behavior of many species of snake, yet trunk function during predatory strikes is poorly understood. Axial kinematics of three thamnophiine snakes (Thamnophis couchii, Thamnophis elegans and Nerodia rhombifer) were studied to quantify differences between sideways-directed and forward-directed attacks and to investigate strike diversity at relatively low phylogenetic levels. Feeding strikes were filmed at 60 Hz, and 13 points along the head and body were digitized. These points were used to calculate body segment displacement, rotation and velocity during predatory strikes. Kinematic analysis revealed significant differences in the foraging modes of these aquatic-feeding species. T. couchii displayed a stereotypical pre-strike posture in which the entire body was arranged in a series of loops directed towards the prey. Forward displacement of body segments sometimes occurred over the entire body in T. couchii but was restricted to the anterior one-third of the trunk in T. elegans and N. rhombifer. T. couchii and N. rhombifer both struck rapidly compared with T. elegans, although N. rhombifer typically had a short strike distance. N. rhombifer struck significantly faster than T. elegans. Aquatic prey capture diversity appears to reflect ecological diversity in thamnophiine snakes.

Animals↗
PubMed1996

Effects of hypoxia and hyperoxia on oxygen-transfer properties of the blood of a viviparous snake

Red cell oxygen affinity, red cell nucleoside triphosphate (NTP) levels and blood oxygen-carrying capacity were determined for male, nonpregnant and pregnant female, and fetal garter snakes Thamnophis elegans exposed to hypoxia (5 % oxygen) and hyperoxia (100 % oxygen). Male and nonpregnant female snakes were maintained under these conditions for up to 3 weeks and exhibited an apparent maximal change in oxygen affinity after 14 days of hypoxia and hyperoxia. Red cell NTP levels decreased and oxygen affinity increased with exposure to hypoxia, while exposure to hyperoxia promoted an increase in red cell NTP concentrations and a decrease in red cell oxygen affinity in the males. Hyperoxia-exposed nonpregnant females did not show a significant change in oxygen affinity. After 14 days of hypoxia, the pregnant females showed an increase in red cell oxygen affinity which was associated with a decrease in red cell NTP concentration and in the molar ratio of NTP/hemoglobin relative to normoxic controls. Fourteen days of hyperoxia did not result in a change in oxygen affinity of red cells from the pregnant female, but did promote a slight increase red cell NTP concentrations. The blood parameters of fetuses from females exposed to hypoxia or hyperoxia did not differ from those of normoxic control fetuses. The fetuses of females exposed to hypoxia suffered greater mortality, appeared less developed and had a lower average wet mass than the fetuses of normoxic- and hyperoxic-exposed females. Neither hypoxia nor hyperoxia altered the oxygen-carrying capacity of the blood in any group of snake.

Journal Article↗
PubMed1998

Characterization of an apoptosis-inducing factor in Habu snake venom as a glycyrrhizin (GL)-binding protein potently inhibited by GL in vitro.

By means of successive heparin-affinity and glycyrrhizin (GL)-affinity column chromatographies (HPLC), a 55 kDa GL-binding protein (gp55) was purified to apparent homogeneity from the Superdex P-I fraction of Habu snake venom. This gp55 was identified as an apoxin I-like protein, because (i) its 20 N-terminal amino acid residues (AHDRNPLEEYFRETDYEEFL) are 95% identical with the corresponding sequence of apoxin I (apoptosis-inducing factor, approx. 55 kDa) in the venom of the western diamondback rattlesnake; and (ii) L-amino acid oxidase (LAO) activity of gp55 is detected when incubated with L-leucine, but not with D-leucine. GL inhibited the LAO activity of gp55 in a dose-dependent manner, but had no effect on the activity of a 65 kDa LAO also purified from Habu snake venom. In addition, GL reduced the ability of gp55 to induce the hemolysis of sheep red blood cells. These results suggest that GL is a potent inhibitor of apoxin I-like proteins in harmful snake venoms.

Amino Acid Oxidoreductases↗
PubMed1997

Histological study of the seminiferous epithelium in the Japanese rat snake, Elaphe climacophora: identification of spermatogonium.

To clarify the features of the seminiferous epithelium of the Japanese rat snake, Elaphe climacophora, the identification of spermatogonium and the examination of features of cell to cell junctions were performed in the present study. As for the identification, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined immunohistochemically to mark spermatogonia. The seminiferous epithelium was observed throughout a year at the electron microscopic level. BrdU immunoreactivity was detected not only in the cells of the first layer of the seminiferous epithelium but also in the second and/or third layers. The cells immunoreactive in the first layer did not seem to attach to the basement membrane and were recognized throughout a year. To investigate cell to cell junctions, we performed actin filament detection by phalloidin staining. Distribution of actin filaments was different from that in mammalian species. At the ultrastructural level, Sertoli-Sertoli cell junctions were observed. Sertoli cells formed junctional complexes. Tight junctions were clearly found, but lacked the backing by actin filaments. These results indicate that the blood-testis barrier of the Japanese rat snake was structurally different from that of mammalian species. In conclusion, the seminiferous epithelium of the Japanese rat snake is intermediate in morphology between amphibians and mammals.

Actins↗
PubMed1998

Contortrostatin, a homodimeric snake venom disintegrin, is a potent inhibitor of osteoclast attachment.

Disintegrins are small disulfide-rich proteins containing an Arg-Gly-Asp (RGD) sequence near their carboxyl terminus. These polypeptides inhibit binding of adhesion molecules to their receptors (integrins) on the surface of cells. Osteoclasts express integrins, heterodimeric cell surface adhesion receptors, that have been shown to be involved in interactions with the extracellular matrix (ECM), including attachment to bone and bone resorption. It has recently been shown that disintegrins effectively inhibit attachment of osteoclasts to components of the ECM and also disrupt osteoclast-mediated bone resorption. Here we characterize the effects of contortrostatin (CTS), a novel homodimeric snake venom disintegrin, on osteoclast attachment. Plastic dishes coated with CTS were able to support osteoclast attachment with a high affinity (EC50,CTS = 86 +/- 6.7 nM) similar to that of vitronectin (VTN; EC50,VTN = 80 +/- 20 nM). Further, CTS was observed to inhibit completely osteoclast attachment to fetal bovine serum (FBS; IC50,FBS = 0.36 +/- 0.04 nM) and VTN (IC50,VTN = 0.85 +/- 0.13 nM). We used monoclonal antibodies directed against the beta1 (monoclonal antibody [MAb] CD29) and beta3 (MAb F11) integrin subunits to explore the mechanism of osteoclast attachment to immobilized CTS. Only MAb F11 inhibited attachment to immobilized CTS (IC50 = 0.41 +/- 0.12 microg/ml), suggesting that binding to CTS is mediated in part by a beta3 integrin, presumably the alpha(v)beta3 VTN receptor. In further support of an integrin-mediated mechanism, binding of osteoclasts to CTS is inhibited by the RGD peptide, GRGDSP. CTS was also more potent (IC50,FBS = 0.36 +/- 0.04 nM) at inhibiting osteoclast attachment to FBS-coated wells than the monomeric snake venom disintegrin echistatin (IC50,FBS = 8.9 +/- 1.5 nM) or VTN (IC50FBS = 97.5 +/- 25.5 nM). Taken together, these data suggest that the snake venom disintegrin CTS is a potent inhibitor of beta3 integrin-mediated osteoclast attachment, presumably involving the VTN receptor (an alpha[v]beta3 integrin). Further studies of the mechanism of CTS-osteoclast interactions may aid in the design of peptide mimetics to act as antiresorptive agents for the treatment of osteoporosis and other skeletal pathology.

Animals↗