Age-related Variation in Snake Venom: Evidence from Two Snakes (Naja atra and Deinagkistrodon acutus) in Southeastern China

In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.

Study on the metabolism of two dominant copepods: Calanoides acutus and Metridia gerlachei collected in summer from the marginal ice zone of the Prydz Bay, Antarctica

Metabolic rates (oxygen consumption and ammonia excretion) of two dominant copepods, Calanoides acutus and Metridia gerlachei, were measured during the austral summer (December 1998 January 1999), in the marginal ice zone of the Prydz Bay. Their elemental compositions (C and N) were also analysed through these metabolic experiments. The results showed that C. acutus was 48.4% 48.6% of dry weight in carbon content and 6.1% 6.5% of dry weight in nitrogen content. Metridia gerlachei was 47.1% 50.1% and 5.9% 7.4%, relatively. Oxygen consumption rates of C. acutus were variant in the different areas (0.226 to 0.774 μlO 2mg -1h -1). And its ammonia excretion rates ranged from 0.014 to 0.105 μgNmg -1h -1. The ratio between the rates (O∶N) exhibited a large difference(5 6 times). For Metridia gerlachei, oxygen consumption rates were 0.488 0.644 μlO 2mg -1 and ammonia excretion rates were 0.015 0.034 μgNmg -1h -1. From the results of metabolic rate measurements and elemental analyses, daily losses of body carbon and nitrogen were estimated to be 0.59% 1.99% and 0.44% 3.27%, respectively. The metabolic carbon requirements accounted for 0 6% 29 2% of primary production per day, and their excretion of ammonia occupied 0.2% 38.0% of daily phytoplankton nitrogen demand.

Increased Efficacy of Antivenom Combined with Hyperbaric Oxygen on Deinagkistrodon acutus Envenomation in Adult Rats

Background：Snakebites are a neglected threat to global human health with a high morbidity rate.The present study explored the efficacy of antivenom with hyperbaric oxygen （HBO） intervention on snakebites,which could provide the experimental basis for clinical adjuvant therapy.Methods：Male Sprague-Dawley rats （n =96） were randomized into four groups：the poison model was established by injecting Deinagkistrodon acutus （D.acutus） venom （0.8 LD50） via the caudal vein;the antivenom group was injected immediately with specific antivenom via the caudal vein after successful establishment of the envenomation model;and the antivenom ＋ HBO group was exposed to HBO environment for 1 h once at predetermined periods of 0 h,4 h,12 h,and 23 h after antivenin administration.Each HBO time point had six rats;the control group was left untreated.The rats in the experimental group were euthanized at the corresponding time points after HBO therapy,and brain tissue and blood were harvested immediately.Hematoxylin and eosin （H＆E） staining was used to investigate the pathological changes in the rat brain.Immunohistochemistry （IHC）,real-time polymerase chain reaction （PCR）,and Western blotting were used to detect the expression ofNestin mRNA and protein in the subventricular zone （SVZ） of the brain.The levels of coagulation function （prothrombin time,activated partial thromboplastin time [APTT],and fibrinogen） and oxidation/antioxidation index （malondialdehyde [MDA] and superoxide dismutase [SOD]） were analyzed.Data were analyzed using one-way analysis of variance.Results：The brain tissue from rats in the poison model was observed for pathological changes using H＆E staining.Tissues showed edema,decreased cell number,and disordered arrangement in the SVZ in the snake venom group.The antivenom-HBO intervention significantly alleviated these observations and was more prominent in the antivenom ＋ HBO group.The serum levels of SOD and MDA in the snake venom group were increased and the antivenom-HBO intervention further increased the SOD levels but significantly decreased the MDA levels;however,this was enhanced within 1 h after HBO administration （MDA：F=5.540,P=0.008,SOD：F=7.361,P =0.000）.Activated partial thromboplastin time （APTT） was significantly abnormal after venom administration but improved after antivenom and was even more significant in the antivenom ＋ HBO group 5 h after envenomation （F =25.430,P =0.000）.Only a few nestin-positive cells were observed in the envenomation model.The expression levels were significant in the antivenom and antivenom ＋ HBO groups within 1 and 5 h after envenomation and were more significant in the antivenom ＋ HBO group as determined by IHC,real-time PCR,and Western blotting （P 〈 0.05）.D.acutus envenomation has neurotoxic effects in the brain of rats.Conclusions：Antivenin and HBO,respectively,induced a neuroprotective effect after D.acutus envenomation by attenuating brain edema,upregulating nestin expression in SVZ,and improving coagulopathy and oxidative stress.The intervention efficacy of antivenom with HBO was maximum within 5 h after envenomation and was more efficacious than antivenom alone.

Purification and characterization of anti-clotting protein component （ACPF-7221） from venom of Agkistrodon acutus

Background Snake venom contains a number of components with different pharmacological and biological activities, especially in cancer therapy, and has increasingly become a research focus. This study was designed to isolate and purify a novel anti-clotting protein component from the venom of Agkistrodon acutus, and to explore its physico-chemical properties and biological activity. Methods The venom of Agkistrodon was isolated and purified by ion-exchange chromatography on diethylaminoethyl （DEAE）-Sepharose Fast Flow, molecular sieve filtration through Sephadex G75, SP-Sepharose Fast Flow and molecular sieve filtration through Sephadex G50. We detected the activated partial thromboplastin time （APTT） of the eluant to select the anti-clotting protein component of interest. The molecular weight was determined by sodium dodecyl sulfate-polyacrylamid gel electrphoresis （SDS-PAGE） and liquid chromatography. Its protein content was detected by bicinchoninic acid （BCA）. Results SDS-PAGE vertical gel electrophoresis showed that the anticoagulant factor is a tripolymer composed of three proteins whose molecular weights are 25 KDa, 30 KDa and 50 KDa. The factor contains about 65% percent protein. Conclusions A novel anti-clotting protein component was purified by ion-exchange chromatography and molecular sieve filtration from the venom of Agkistrodon acutus and was found to be composed of three kinds of proteins.

鱼腥草地上部分和根部提取物抑制尖吻蝮蛇毒的比较

研究鱼腥草Houttuynia cordata Thunb地上部分提取物和根部提取物对尖吻蝮Deinagkistrodon acutus蛇毒抑制作用。采用体外实验检测两种提取物对尖吻蝮蛇毒中主要酶类(蛋白水解酶、磷脂酶A2(PLA 2)、透明质酸酶等)的抑制作用;采用体内实验检测其对尖吻蝮蛇毒诱导体内毒性(出血、水肿、组织坏死和致死毒性)的抑制作用。鱼腥草地上部分提取物对尖吻蝮蛇毒蛋白水解酶、磷脂酶A2和透明质酸酶活性具有显著的抑制作用,同时能有效抑制促凝活性和纤维蛋白原水解活性;在体内,能够使尖吻蝮蛇毒引起的出血、水肿、组织坏死和致死毒性明显减弱。相比之下,鱼腥草根部提取物也表现出类似的抑制模式,但在相同浓度下其抑制效果均低于地上部分提取物。植物化学成分分析表明,两种提取物中均含有生物碱、蒽醌类物质和蛋白质;地上部分提取物中含有黄酮、多酚、单宁、三萜、甾醇和皂苷类物质,而根部提取物中缺少这些物质。实验结果首次验证了鱼腥草地上部分及根部对尖吻蝮蛇毒的抑制作用,并证实地上部分比根部在抑制毒液的主要酶类和体内毒性方面更为有效,表明鱼腥草地上部分可以作为一种潜在资源用于开发治疗毒蛇咬伤的解毒剂。

Proline as a biochemical marker in relation to the ecology of two halophytic Juncus species

Aims osmolytes,used for maintaining osmotic balance and as‘osmoprotectants’,are synthesized in plants as a general,con-served response to abiotic stress,although their contribution to stress-tolerance mechanisms remains unclear.Proline,the most common osmolyte,accumulates in many plant species in parallel with increased external salinity and is considered a reliable bio-chemical marker of salt stress.We have measured proline levels in two halophytic,closely related Juncus species under laboratory and field conditions to assess the possible relevance of proline biosynthesis for salt tolerance and therefore for the ecology of these two taxa.Methods Proline was quantified in plants treated with increasing NaCl con-centrations and in plants sampled in two salt marshes located in the provinces of Valencia and alicante,respectively,in southeast spain.Electrical conductivity,pH,Na+and Cl−concentrations were measured in soil samples collected in parallel with the plant material.Important Findings Treatment with NaCl inhibited growth of J.acutus plants in a concentration-dependent manner,but only under high salt conditions for J.maritimus.salt treatments led to proline accumulation in both species,especially in the more salt-tolerant J.maritimus.The results,obtained under laboratory conditions,were confirmed in plants sam-pled in the field.in all the samplings,proline contents were signifi-cantly lower in J.acutus than in the more tolerant J.maritimus growing in the same area.No direct correlation between soil salinity and proline levels could be established,but seasonal variations were detected,with increased proline contents under accentuated water deficit conditions.our results suggest that proline biosynthesis is not only an induced,general response to salt stress but also an important contributing factor in the physiological mechanisms of salt tolerance in Juncus,and that it therefore correlates with the ecology of both species.