Early decrease of ionized calcium and static symmetric dimethylarginine concentration in a model of renal tubular necrosis in corn snakes (Pantherophis guttatus).

OBJECTIVE: To determine if plasma concentrations of symmetric dimethylarginine (SDMA), N-acetyl-beta-d-glucosaminidase (NAG), GGT, ALT, AST, lactate, total calcium, and ionized calcium (iCa) and the calcium:phosphorus ratio are clinically relevant biomarkers to detect early stages of tubular lesions in snakes. ANIMALS: 6 adult corn snakes (Pantherophis guttatus). METHODS: Corn snakes were administered 11 injections of gentamicin at 50 mg/kg, SC, q 24 h in an experimental model of induced tubular necrosis. Plasma biochemistry and blood gas analyses were performed at baseline and after the 3rd and 11th injections. Parameters were compared between time points using a paired Wilcoxon test. In 3 individuals, renal biopsies were collected at baseline before starting injections and at the 3rd and 11th injections, while renal tissue samples were procured after euthanasia in all individuals. RESULTS: Renal proximal and distal tubular necrosis and hepatic steatosis were present in all individuals at necropsy. Compared to baseline, decreased blood concentrations of lactate, ionized calcium, and total calcium and a decreased calcium:phosphorus ratio were noted. A significant decrease of lactate and ionized calcium was observed after 3 days. Conversely, no changes in SDMA, NAG, ALT, AST, GGT, and sodium were detected. CLINICAL RELEVANCE: Ionized calcium and lactate concentrations were the earliest parameters to decrease compared to baseline values in this experimental model. While SDMA is a sensitive indicator of renal disease in mammals, this biomarker did not increase in a model of induced acute tubular necrosis in corn snakes.

Disparities in opioid use disorder-related hospital use among postpartum Virginia Medicaid members.

INTRODUCTION: The overdose crisis is increasingly revealing disparities in opioid use disorder (OUD) outcomes by race and ethnicity. Virginia, like other states, has witnessed drastic increases in overdose deaths. However, research has not described how the overdose crisis has impacted pregnant and postpartum Virginians. We report the prevalence of OUD-related hospital use during the first year postpartum among Virginia Medicaid members in the years preceding the COVID-19 pandemic. We secondarily assess how prenatal OUD treatment is associated with postpartum OUD-related hospital use. METHODS: This population-level retrospective cohort study used Virginia Medicaid claims data for live infant deliveries between July 2016 and June 2019. The primary outcome of OUD-related hospital use included overdose events, emergency department visits, and acute inpatient stays. Independent variables of interest were prenatal receipt of medication for OUD (MOUD) and receipt of non-MOUD treatment components in line with a comprehensive care approach (e.g., case management, behavioral health). Both descriptive and multivariate analyses were performed for all deliveries and stratified by White and Black non-Hispanic individuals to bring attention to the devastating impacts of the overdose crisis within communities of color. RESULTS: The study sample included 96,649 deliveries. Over a third were by Black birthing individuals (n = 34,283). Prenatally, 2.5 % had evidence of OUD, which occurred more often among White (4 %) than Black (0.8 %) non-Hispanic birthing individuals. Postpartum OUD-related hospital use occurred in 10.7 % of deliveries with OUD, more commonly after deliveries by Black, non-Hispanic birthing individuals with OUD (16.5 %) than their White, non-Hispanic counterparts (9.7 %), and this disparity persisted in the multivariable analysis (Black AOR 1.64, 95 % CI 1.14-2.36). Postpartum OUD-related hospital events were less frequent for individuals receiving versus not receiving postpartum MOUD within 30 days prior to the event. Prenatal OUD treatment, including MOUD, was not associated with decreased odds of postpartum OUD-related hospital use in the race-stratified models. CONCLUSION: Postpartum individuals with OUD are at high risk for mortality and morbidity, especially Black individuals not receiving MOUD after delivery. An urgent need remains to effectively address the systemic and structural drivers of racial disparities in transitions of OUD care through the one-year postpartum period.

Neuronal plasticity in the forebrain of the male red-sided garter snake: Effect of season, low temperature dormancy, and hormonal status on dendritic spine density.

Numerous studies have reported seasonal variations in regional morphology in the brains of seasonally breeding vertebrates. These alterations of neuronal morphology and dendritic spine density appear to be an active process within specific brain nuclei that regulate seasonal behaviors. In many cases, this neural plasticity has been found to be in response to changes in circulating sex steroid hormone levels and occur within pathways essential for the control of reproductive behaviors. Male red-sided garter snakes (Thamnophis sirtalis parietalis) (RSGS) exhibit a dissociated reproductive pattern where mating is initiated at a time when the gonads and steroidogenesis are inactive. And, although circulating levels of sex steroid hormones are elevated at the initiation of courtship and mating, the only known cue found to initiate courtship behavior and mating, is an extended period of low temperature dormancy (LTD) followed by exposure to warm temperatures. This study was designed to examine the role of seasons, sex steroid hormones, and LTD on neuronal and dendritic spine density within the anterior hypothalamus-preoptic area (AHPOA), a region shown to be critical for the regulation of reproductive behaviors. In the male RSGS, the density of dendritic spines on neurons in the AHPOA was significantly greater in spring, actively courting animals, than summer or fall, non-courting animals. Animals maintained under conditions of LTD exhibited significantly increasing spine density as time maintained in LTD increased. Animals receiving either testosterone or estradiol had a significantly greater density of dendritic spines than control animals. This study offers evidence suggesting that the "set up" of the pathways controlling courtship behavior and mating in the male RSGS, is not due solely to an extended period of LTD, but that an extended period of LTD in conjunction with circulating sex steroid hormones are critical for the initiation of reproductive behavior.

Development and evaluation of a PCR-based assay kit for authentication of Zaocys dhumnades in traditional Chinese medicine.

We developed a kind of Zaocys dhumnades DNA test kit and it's indexes including specificity, sensitivity and stability were evaluated and compared with the method recorded in Chinese Pharmacopoeia (2010 edition). The bioinformatics technology was used to design primers, sequencing and blast, in conjunction with PCR technology based on the characteristics of Z. dhumnades cytochrome b (Cyt b) gene. The efficiency of nucleic acid extraction by the kit was done in accordance with Pharmacopoeia method. The kit stability results proved effective after repeated freezing and thawing 20 times. The sensitivity results indicated that the lowest amount detected by the kit was 0. 025 g of each specimen. The specificity test of the kit was 100% specific. All repeatability tests indicated the same results when conducted three times. Compared with the method recorded in Chinese Pharmacopoeia, the PCR-based assay kit by our team developed is accurate, effective in identification of Z. dhumnades, it is simple and fast, demonstrating a broad prospect in quality inspection of Z. dhumnades in the future.

Edema and Nociception Induced by Philodryas patagoniensis Venom in Mice: A Pharmacological Evaluation with Implications for the Accident Treatment.

We have investigated the mechanisms involved in the genesis of edema and nociception induced by Philodryas patagoniensis venom (PpV) injected into the footpad of mice. PpV induced dose-related edema and nociceptive effects. Pretreatment of mice with cyclooxygenase inhibitor (indomethacin), but not with cyclooxygenase 2 inhibitor (celecoxib) markedly inhibited both effects. Pretreatments with H1 receptor antagonist (promethazine) or with dual histamine-serotonin inhibitor (cyproheptadine) failed in inhibiting both effects. In groups pretreated with captopril (angiotensin-converting enzyme inhibitor) the edema was unaltered, but nociception was clearly increased, suggesting the participation of kinins in the pathophysiology of the nociception but not of the edema-forming effect of PpV. When PpV was treated with EDTA, the nociception was similar to the one induced by untreated venom, but edema was markedly reduced. We concluded that PpV-induced edema and nociception have cyclooxygenase eicosanoids as the main mediators and no participation of vasoactive amines. Kinins seem to participate in nociception but not in edema induced by PpV. The results also suggest that metalloproteinases are the main compounds responsible for the edema, but not for the nociception induced by this venom.

Hydrostatin-TL1, an Anti-Inflammatory Active Peptide from the Venom Gland of Hydrophis cyanocinctus in the South China Sea.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine with intense pro-inflammatory and immunomodulatory properties, and anti-TNF-α biologics are effective therapies for various inflammatory diseases such as inflammatory bowel disease (IBD) and sepsis. Snake venom, as a traditional Chinese medicine, has been used in the treatment of inflammatory diseases in China for centuries. In this research, we constructed a venom gland T7 phage display library of the sea snake Hydrophis cyanocinctus to screen bioactive compounds that antagonize TNF-α and identified a novel nine-amino-acid peptide, termed hydrostatin-TL1 (H-TL1). In enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analyses, H-TL1 inhibited the interaction between TNF-α and TNF receptor 1 (TNFR1). Further, H-TL1 attenuated the cytotoxicity of TNF-α in L929 cells as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. H-TL1 also decreased the mRNA expression of TNF-α/TNFR1 downstream targets and suppressed the phosphorylation of well-characterized proteins of downstream signal transduction pathways in HEK-293 cells. In vivo data demonstrated that H-TL1 protects animals against dextran sodium sulfate (DSS)-induced acute colitis and lipopolysaccharide (LPS)-induced acute shock. Given its significant anti-inflammatory activity in vitro and in vivo, H-TL1 is a potential peptide for the development of new agents to treat TNF-α-associated inflammatory diseases.

Seasonal and sex differences in responsiveness to adrenocorticotropic hormone contribute to stress response plasticity in red-sided garter snakes (Thamnophis sirtalis parietalis).

As in many vertebrates, hormonal responses to stress vary seasonally in red-sided garter snakes (Thamnophis sirtalis parietalis). For example, males generally exhibit reduced glucocorticoid responses to a standard stressor during the spring mating season. We asked whether variation in adrenal sensitivity to adrenocorticotropic hormone (ACTH) explains why glucocorticoid responses to capture stress vary with sex, season and body condition in red-sided garter snakes. We measured glucocorticoids at 0, 1 and 4 h after injection with ACTH (0.1 IU g(-1)body mass) or vehicle in males and females during the spring mating season and autumn pre-hibernation period. Because elevated glucocorticoids can influence sex steroids, we also examined androgen and estradiol responses to ACTH. ACTH treatment increased glucocorticoids in both sexes and seasons. Spring-collected males had a smaller integrated glucocorticoid response to ACTH than autumn-collected males. The integrated glucocorticoid response to ACTH differed with sex during the spring, with males having a smaller glucocorticoid response than females. Although integrated glucocorticoid responses to ACTH did not vary with body condition, we observed an interaction among season, sex and body condition. In males, ACTH treatment did not alter androgen levels in either season, but androgen levels decreased during the sampling period. Similar to previous studies, plasma estradiol was low or undetectable during the spring and autumn, and therefore any effect of ACTH treatment on estradiol could not be determined. These data provide support for a mechanism that partly explains how the hypothalamus-pituitary-adrenal (HPA) axis integrates information about season, sex and body condition: namely, variation in adrenal responsiveness to ACTH.

Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom.

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, we examined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protect against the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract caused moderate twitch-tension facilitation at low concentrations (107.4 ± 6.2% with 20 µl/ml and 118.9 ± 9.3% with 40 µl/ml in PND, and 120.7 ± 7.7% with 40 µl/ml and 114.5 ± 4.4% with 50 µl/ml in BC after 120 min; n = 4-6, mean ± SEM). In PND, the ethanol alone (40 µl/ml, n = 4) did not change the twitch-tension when compared with control. However, in BC, the ethanol produced a higher facilitation when compared to control. At higher concentrations (>50 µl/ml) the extract caused total and reversible blockade in both preparations. Venom (50 µg/ml) caused partial blockade in PND (58.5 ± 12%, n = 4) and almost total blockade in BC (93.5 ± 2.2%, n = 4). Pretreatment of the preparations with extract (40 µl/ml) for 30 min before incubation with venom (50 µg/ml) completely protected PND from neuromuscular blockade and delayed the blockade in BC. The extract alone caused only mild morphological alterations (12.5 ± 0.5% and 10.9 ± 2.3% fiber damage in PND and BC, respectively, compared to 2.3 ± 0.3% and 3 ± 0 in controls; n = 3), with no increase in expression of the inflammatory cytokines TNFα and IFNγ. The ethanol alone also caused slight muscle damage: 4.3 ± 2.4% in PND and 6.7 ± 3.3% in BC (both n = 3) and little or no TNFα and IFNγ expression in both preparations as observed in control. Venom (50 µg/ml) caused 53.5 ± 8.5% and 55.8 ± 4.3% fiber damage in PND and BC, respectively; (n = 3, p < 0.05 vs. controls) and enhanced expression of TNFα and IFNγ. Pretreatment of the preparations with extract protected against venom-induced muscle damage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFα and IFNγ expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.

Embryonic mobilization of calcium in a viviparous reptile: evidence for a novel pattern of placental calcium secretion.

Yolk reserves supply the majority of embryonic nutrition in squamate reptiles, including calcium. Embryos of oviparous squamates exploit the eggshell for supplemental calcium, while embryos of viviparous species may receive additional calcium via the placenta. Developmental uptake of calcium in oviparous snakes increases during the interval of greatest embryonic growth (stage 35 to parturition). However, the pattern of embryonic calcium acquisition is unknown for viviparous snakes. Furthermore, while the uterus of oviparous species transports calcium early in embryonic development during mineralization of the eggshell, the timing of uterine calcium secretion in viviparous snakes is unknown. We studied a viviparous snake, Virginia striatula, to determine the ontogenetic pattern of yolk and embryonic calcium content. The pattern of embryonic calcium uptake of V. striatula is similar to that of oviparous snakes but the sources of calcium differ. In contrast to oviparous species, embryos of V. striatula acquire half of total neonatal calcium via placental provision, of which 71% is mobilized between stage 35 and parturition. Furthermore, we report for the first time in a viviparous squamate an increase in yolk calcium content during early stages of embryonic development, indicating that uterine secretion of calcium occurs in V. striatula coincident with shelling in oviparous squamates. Thus, uterine calcium secretion in this viviparous species may either occur continuously or in two phases, coincident with the timing of shelling in oviparous species and again during the last stages of development. Whereas, the pattern of embryonic calcium acquisition in V. striatula is plesiomorphic for squamates, the pattern of uterine calcium secretion includes both retention of a plesiomorphic trait and the evolution of a novel trait.

[Effect of soluble total proteins of Zaocys dumnades on interleukin-1beta, tumor necrosis factor-alpha and interleukin-10 expressions in human fibroblast-like synoviocytes in vitro].

OBJECTIVE: To evaluate the effect of soluble total proteins of Zaocys dhumnades on the expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and IL-10 in human fibroblast-like synoviocytes (FLS) cultured in vitro. METHODS: Primary cultured FLS isolated from the synovium of patients with rheumatoid arthritis (RA) were incubated in the presence of different concentrations (50, 150 and 450 microg/ml) of soluble total proteins of Zaocys dhumnades, with Tripterygium hypoglaucum Hutch (THH) and DMEM as the positive and negative controls, respectively. Enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to detect the expressions of IL-1beta, IL-10 and TNF-alpha in the FLS. RESULTS: The protein and mRNA levels of IL-1beta and TNF-alpha in the supernatant of the FLS exposed to 150 and 450 microg/ml of the soluble total proteins of Zaocys dhumnades decreased, while IL-10 protein and mRNA increased significantly as compared with those in the negative control group (P<0.01). CONCLUSION: The soluble total proteins of Zaocys dhumnades produce therapeutic effect on RA possibly by inhibiting IL-1beta and TNF-alpha and promoting IL-10 expressions in the FLS.

[The effect of the type II collagen protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis].

OBJECTIVE: To investigate the effect of the type II collagen (C II) protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis (AA). METHODS: Type II collagen protein was abstracted and purificated from Zaocys. Adjuvant arthritis (AA) was induced by a single intradermal injection of 0.1 mL of complete Freund's adjuvant into the left hind paw. Synoviocytes' supernatants were harvested and synoviocytes-Peyer's Patches (PP) cells coculture system were developed. Tumor necrosis factor-alpha (TNF-alpha) activity was measured by L929 cytotoxicity bioassay and Interleukin (IL)-1beta activity was measured by MTT dye reduction. The synoviocytes' supernatants cytokines' levels were detected by ELISA. RESULTS: Each concentration of C II from Zaocys had no effect on IL-1beta and TNF-alpha production by synoviocytes in vitro. Middle concentration of C II suppressed the activity of IL-1beta and TNF-alpha production by synoviocytes-PP cells coculture system (P < 0.05). Treating with low and high dose of C II suppressed the activity of TNF-alpha and IL-1beta producing by synoviocyte (P < 0.05), significantly suppressed in the group of AA rats treated with middle dose of C II (P < 0.01). Treating with middle and high dose of C II decreased the level of synoviocytes' supernatants TNF-alpha (P < 0.05), the level of synoviocytes' supernatants IL-1beta decreased in all treating groups (P < 0.05). Treating with middle dose of C II increased the level of serum TGF-beta (P < 0.05). Middle concentration of C II suppressed the activity of IL-1 and TNF production by synoviocytes-PP cells coculture system (P < 0.05). CONCLUSIONS: C II from Zaocys has no direct effect on the activity of IL-1beta and TNF production by synoviocytes in vitro. Oral administration of type II collagen protein from Zaocys can effectively suppressed the activity and level of the cytokines production by synoviocytes in rats with adjuvant arthritis (AA).

Effect of soluble total proteins of Zaocys dumnades on interleukin-1beta, tumor necrosis factor-alpha and interleukin-10 expressions in human fibroblast-like synoviocytes in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of soluble total proteins of Zaocys dhumnades on the expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and IL-10 in human fibroblast-like synoviocytes (FLS) cultured in vitro.</p><p><b>METHODS</b>Primary cultured FLS isolated from the synovium of patients with rheumatoid arthritis (RA) were incubated in the presence of different concentrations (50, 150 and 450 microg/ml) of soluble total proteins of Zaocys dhumnades, with Tripterygium hypoglaucum Hutch (THH) and DMEM as the positive and negative controls, respectively. Enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to detect the expressions of IL-1beta, IL-10 and TNF-alpha in the FLS.</p><p><b>RESULTS</b>The protein and mRNA levels of IL-1beta and TNF-alpha in the supernatant of the FLS exposed to 150 and 450 microg/ml of the soluble total proteins of Zaocys dhumnades decreased, while IL-10 protein and mRNA increased significantly as compared with those in the negative control group (P<0.01).</p><p><b>CONCLUSION</b>The soluble total proteins of Zaocys dhumnades produce therapeutic effect on RA possibly by inhibiting IL-1beta and TNF-alpha and promoting IL-10 expressions in the FLS.</p>

[Effect of Xianlong granules on immunological function in rats of adjuvant arthritis].

OBJECTIVE: To study the effect of Xianlong granules (XLG) on immunological function in the rat of adjuvant arthritis (AA). METHOD: Rats were randomly divided into normal group, AA model group, prednisone group and low, middle and high dose XLG groups, 10 rats in each group. All rats were treated by intragastric administration from the 18 days after arthritis was induced by the complete Freud's adjuvant and the effect of XLG on toes swelling was observed. On the 30th days after modeling, proliferation of the splenic and thymic lymphocytes, and IgG secreted by splenocytes were detected respectively by MTT assay and ELISA. RESULT: Compared with the model group, both the high and middle dose XLG groups had significant therapeutic effects on toes dwelling in the rat of AA (P < 0.05 or P < 0.01); The low, middle and high dose XLG groups strengthened the PHAM-inhibited proliferation of splenic lymphocytes (P < 0.05), and inhibited the PHAM-augmented proliferation of thymic lymphocytes (P < 0.05); XLG did not significantly effect on IgG level secreted by splenocytes in rats of AA. CONCLUSION: XLG can cure toes swelling in rats of AA, which is related with regulation of the abnormal immunlological function.

Metal levels in blood, muscle and liver of water snakes (Nerodia spp.) from New Jersey, Tennessee and South Carolina.

Reptiles, particularly snakes, could serve as bioindicators of contamination because some are comparatively long-lived, exhibit different trophic levels, and are at the top of their food chains. We test the null hypothesis that there are no differences in the concentrations of heavy metals in the blood, muscle and liver of water snakes (Nerodia spp.) from rivers in New Jersey, Tennessee and South Carolina. While the former site is in an urban/suburban area, the latter two sites are relatively rural and are located on Department of Energy sites. For the snakes from New Jersey, there were significant differences in metal concentrations among tissues for all metals, the highest levels for arsenic and selenium were in liver and kidney, for cadmium were in the liver, for chromium and lead were in skin, and for mercury and manganese were in the muscle. Body length was not correlated with metal levels, and there were more significant correlations for skin with internal tissues than for blood with other tissues. There were more significant correlations for mercury than for other metals. In comparing metal levels among states, levels were generally higher for snakes collected from South Carolina. These data indicate that, since water snakes accumulate contaminants differentially as a function of location, they can be useful bioindicators of environmental exposure to contaminants. Moreover, because of their wide geographical distribution and use of varying trophic compartments, this genus can be useful for cross-site comparisons.

Effect of Xianlong granules on immunological function in rats of adjuvant arthritis / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Xianlong granules (XLG) on immunological function in the rat of adjuvant arthritis (AA).</p><p><b>METHOD</b>Rats were randomly divided into normal group, AA model group, prednisone group and low, middle and high dose XLG groups, 10 rats in each group. All rats were treated by intragastric administration from the 18 days after arthritis was induced by the complete Freud's adjuvant and the effect of XLG on toes swelling was observed. On the 30th days after modeling, proliferation of the splenic and thymic lymphocytes, and IgG secreted by splenocytes were detected respectively by MTT assay and ELISA.</p><p><b>RESULT</b>Compared with the model group, both the high and middle dose XLG groups had significant therapeutic effects on toes dwelling in the rat of AA (P < 0.05 or P < 0.01); The low, middle and high dose XLG groups strengthened the PHAM-inhibited proliferation of splenic lymphocytes (P < 0.05), and inhibited the PHAM-augmented proliferation of thymic lymphocytes (P < 0.05); XLG did not significantly effect on IgG level secreted by splenocytes in rats of AA.</p><p><b>CONCLUSION</b>XLG can cure toes swelling in rats of AA, which is related with regulation of the abnormal immunlological function.</p>

Snake venom glutaminyl cyclase.

Glutaminyl cyclase (QC) catalyzes N-terminal glutamine cyclization of many endocrine peptides and is typically abundant in brain tissue. As three-finger toxins in the venoms of colubrid snakes Boiga dendrophila and Boiga irregularis contain N-terminal pyroglutamate, we searched for QC in venom glands of both snakes. Here we report cDNA sequences of QC from brain and venom gland tissues of Boiga species. We propose that QC expressed in snake venom gland tissue plays a role in the N-terminal pyroglutamate formation of several snake venom toxins, indirectly contributing to venom potency.

A serotonin receptor antagonist, but not melatonin, modulates hormonal responses to capture stress in two populations of garter snakes (Thamnophis sirtalis parietalis and Thamnophis sirtalis concinnus).

Hormonal and behavioral responses to a stressor depend on many factors, including the influence of other hormones. We examined the role of melatonin in modulating hormonal responses to capture stress in two populations of male garter snakes, Thamnophis sirtalis. Studies of red-sided (T. sirtalis parietalis) and red-spotted (T. sirtalis concinnus) garter snakes were conducted in the field with free-living snakes. Populations of red-sided garter snakes in south-central Manitoba, Canada undergo a period of winter dormancy for approximately 8 months each year followed by an attenuated mating season (4-5 weeks) in early spring. In contrast, the mid-latitude red-spotted garter snake in western Oregon, USA has an extended breeding season and can be active during 10-12 months of the year given appropriate environmental conditions. We chose to study these two populations of garter snakes to investigate possible variation in melatonin function among snakes with different suites of environmental adaptations. To better address these questions, we also examined the effects of 5-hydroxytryptophan (a precursor of melatonin synthesis) and ketanserin (a serotonergic type 2A receptor antagonist) on hormonal responses to capture stress. We observed a trend of increased corticosterone and decreased androgen concentrations in northern-latitude red-sided garter snakes (T. sirtalis parietalis) subjected to 4 h of capture stress during the spring. However, these differences were not statistically significant. During the fall, red-sided garter snakes showed no change in corticosterone or androgen concentrations in response to the capture stress treatments. We speculate that northern-latitude red-sided garter snakes suppress hormonal responses to capture stress during preparation for winter dormancy. Treatment with melatonin, 5-hydroxytryptophan, or ketanserin did not significantly influence corticosterone or androgen concentrations of northern-latitude red-sided garter snakes during the spring or fall. Mid-latitude red-spotted garter snakes (T. sirtalis concinnus) from Oregon showed a statistically significant increase in corticosterone concentrations in response to 4 h of capture stress; treatment with melatonin, 5-hydroxytryptophan, or ketanserin prior to capture stress had no significant influence on plasma corticosterone concentrations. Androgen concentrations of mid-latitude red-spotted garter snakes in response to capture stress were significantly lower than those of non-stressed control snakes. Neither melatonin nor 5-hydroxytryptophan influenced the change in androgen concentrations during capture stress. However, androgen concentrations of snakes treated with ketanserin prior to 4 h of capture stress did not differ significantly from those of non-stressed control snakes. These studies suggest that melatonin does not modulate hormonal responses to capture stress in this ectothermic model. Our results also suggest that a serotonin-regulated system may play a role in modulating the activity of the hypothalamic-pituitary-gonadal axis during physiological stress responses.

Trophic and maternal transfer of selenium in brown house snakes (Lamprophis fuliginosus).

Excessive concentrations of dietary Se are toxic to oviparous vertebrates (i.e., fish and birds) but little is known about its accumulation and effects in reptiles. We exposed female brown house snakes, Lamprophis fuliginosus, to 10 and 20 microg/g Se by injecting seleno-D,L-methionine into their prey items and compared the snakes to individuals receiving background levels of approximately 1 microg/g dietary Se. Snakes were fed meals equaling 25% of their body mass 2-3 times a month for 10 months. Snakes exposed to excessive Se accumulated significant concentrations of Se in kidney, liver, and ovarian tissue, but accumulation had no effect on female survival, food consumption, growth, or body condition. Fewer females exposed to excessive Se reproduced than females exposed to 1 microg/g Se (67% vs. 91%, respectively), but the reduction in reproductive activity was not statistically significant. Total reproductive output of females did not differ among the three dietary treatments. However, snakes exposed to 10 and 20 microg/g Se transferred significant concentrations of Se to their eggs. In the 20 microg/g treatment, maternal transfer resulted in Se concentrations in eggs that surpassed all suggested reproductive toxicity thresholds for birds and fish. Further studies are needed to more rigorously determine whether maternal transfer of Se in this snake species affects the viability of developing embryos or the health of offspring.

Laser ablation-ICP-MS analysis of dissected tissue: a conservation-minded approach to assessing contaminant exposure.

Minimally invasive sampling techniques are an essential ecotoxicological tool for continuous assessment of contaminant exposure and in instances where it is not desirable or practical to sacrifice the animal. In this paper, we report on the application of laser ablation-ICP-MS (LA-ICP-MS) for sampling of minute (approximately 1 mg, 2-3 mm) tail clips of the banded water snake, Nerodia fasciata, as a means to assess contaminant exposure. The snakes were split into three treatments (n = 8) and were fed three increasing levels (control, medium, and high) of fish contaminated with As, Se, and Sr for 24 months. LA-ICP-MS concentrations of tail clips for all three elements were significantly correlated with the remaining whole tail concentration determined by homogenization, acid digestion, and ICP-MS analysis. Additionally, LA-ICP-MS concentrations for As and Se in the tail clip were similar to the acid digestion solution analysis values for the whole tail, which suggests that these elements are homogeneously distributed. Strontium concentrations were underestimated by LA-ICP-MS when compared to whole tail concentrations. Statistical analysis showed that LA-ICP-MS tail clip concentrations differed significantly according to dietary treatment. Posterior probability error rates from nonparametric discriminant function analysis indicated that LA-ICP-MS analysis of tail clips was useful for predicting exposure to Se with only a 4% probability of misclassification among treatments. Errors associated with misclassification of As were greater (17%) but this was, in part, related to the low concentrations of As in the tail (<1 ppm for the highest treatment). Taken together, the findings from this study suggest that LA-ICP-MS of microdissected tissue shows promise as a nondestructive technique for conservation-minded ecotoxicological studies.

Molecular cloning and characterization of protein phosphatase 2C of vomeronasal sensory epithelium of garter snakes.

The earthworm-derived chemoattractant ES20 interacts with its G-protein-coupled receptors on the plasma membrane of vomeronasal (VN) sensory neurons of garter snakes, resulting in an increase in inositol trisphosphate [J. Biol. Chem. 269 (1994) 16867] and a rapid phosphorylation of the membrane-bound proteins, p42/44 [Biochim. Biophys. Acta 1450 (1999) 320]. The phosphorylation of p42/44 proteins are countervailingly regulated by a protein kinase and an okadaic acid-insensitive but fluoride-sensitive protein phosphatase (PPase) [J. Liu et al. (loc. cit.)]. The phosphorylation of p42/44 induced by ES20 appears to play a role in the regulation of signal transduction pathways by modulating the GTPase activity [J. Liu et al. (loc. cit.)]. A 564-bp fragment of cDNA was obtained from VN RNA of garter snakes by reverse transcription polymerase chain reaction with degenerate primers. The 564-bp fragment was amplified, cloned, and sequenced. Northern blot analysis revealed that both the VN organ (VNO) and brain contained the gene of PPase 2C. A full-length complementary 4119-bp DNA containing an open reading frame of 1146bp that encodes a protein of 382 amino acids with a molecular mass of 49,123Da was obtained from the VN cDNA library of garter snakes. The deduced amino acid sequence showed 88% amino acid identity to bovine protein phosphatase 2C alpha and 87% identity to human and rat PP2C alpha and to Mg(2+)-dependent protein phosphatase 1A of rat and rabbit. In situ hybridization revealed that the mRNA of VN protein phosphatase 2C is expressed in the vomeronasal sensory epithelium. This is the first report of the identification of a type 2C serine/threonine protein phosphatase in the VN system.