Applications of snake venoms in treatment of cancer

Snake venoms are folk medicines used since ages. The components of snake venoms have high specific affinity and actions on cells and cell components. Also snake venoms are largely cytotoxic to tumor cells than normal cells. In addition to these, they have several therapeutic actions that make them an attractive option in the management of cancer. The advent of modern technologies has greatly helped in extracting and identifying new components of therapeutic interests in short time. The article highlights the importance of snake venoms in the management of cancer, so as to motivate curious researchers to devote their skills in this fascinating area. This in turn may bring hope, smile and relief to several cancer patients in future.

Comparative study of the hemolytic and cytotoxic activities of nematocyst venoms from the jellyfish Cyanea nozakii Kishinouye and Nemopilema nomurai Kishinouye

Two species of jellyfish, Cyanea nozakii Kishinouye and Nemopilema nomurai Kishinouye, have occurred offcoastal areas of the northeastern China Sea, Yellow Sea, and Bohai Sea in recent years. They influence marine ecosystem safety and fishery production, and also pose a risk to human health. The current study examined the hemolytic and cytotoxic activities of crude venoms extracted from the nematocysts of C. nozakii and N. nomurai. The results showed that there were more nematocysts on tentacles from C. nozakii than on tentacles of the same length from N. nomurai. The protein concentration per nematocyst extracted from N. nomurai was higher than that from C. nozakii. Both nematocyst venoms showed dose-and timedependent hemolytic activity on erythrocytes from chicken, pigeon, and sheep, with sheep erythrocytes being the most sensitive, with EC 50 values of 69.69 and 63.62 μg/m L over a 30-min exposure with N. nomurai and C. nozakii nematocyst venoms, respectively. A cytotoxic assay of both jellyfish venoms on A431 human epidermal carcinoma cells resulted in IC 50 values of 68.6 and 40.9 μg/mL after 24-h incubation, respectively, with venom from C. nozakii showing stronger cytotoxic activity than that from N. nomurai. The results of current study indicate that nematocyst venom from C. nozakii had stronger hemolytic and cytotoxic activities than that from N. nomurai and, thus, C. nozakii might be more harmful to the health of humans and other species than are N. nomurai when they appear in coastal waters.

Two novel antimicrobial peptides from skin venoms of spadefoot toad Megophrys minor

Amphibian skin contains rich bioactive peptides. Especially, a large amount of antimicrobial peptides have been identified from amphibian skin secretions. Antimicrobial peptides display potent cytolytic activities against a range of pathogenic bacteria and fungi and play important defense roles. No antimicrobial peptides have been reported from toads belonging to the family of Pelobatidae. In this work, two novel antimicrobial peptides(Megin 1 and Megin 2) were purified and characterized from the skin venoms of spadefoot toad Megophrys minor(Pelobatidae, Anura, Amphibia). Megin 1 had an amino acid sequence of FLKGCWTKWYSLKPKCPF-NH2, which was composed of 18 amino acid residues and contained an intra-molecular disulfide bridge and an amidated C-terminus. Megin 2 had an amino acid sequence of FFVLKFLLKWAGKVGLEHLACKFKNWC, which was composed of 27 amino acid residues and contained an intra-molecular disulfide bridge. Both Megin 1 and Megin 2 showed potential antimicrobial abilities against bacteria and fungi. The MICs of Megin 1 against Escherichia coli, Bacillus dysenteriae, Staphylococcus aureus, Bacillus subtilis, and Candida albicans were 25, 3, 6.25, 3, and 50 μg·m L^(-1), respectively. The corresponding MICs for Megin 2 were 6.25, 1.5, 12.5, 1.5, and 12.5 μg·m L^(-1), respectively. They also exerted strong hemolytic activity against human and rabbit red cells. The results suggested that megin peptides in the toad skin of M. minor displayed toxic effects on both eukaryotes and prokaryotes. This was the first report of antimicrobial peptides from amphibians belonging to the family of Pelobatidae.

Bioactive peptides from scorpion venoms:therapeutic scaffolds and pharmacological tools

Evolution and natural selection have endowed animal venoms,including scorpion venoms,with a wide range of pharmacological properties.Consequently,scorpions,their venoms,and/or their body parts have been used since time immemorial in traditional medicines,especially in Africa and Asia.With respect to their pharmacological potential,bioactive peptides from scorpion venoms have become an important source of scientific research.With the rapid increase in the characterization of various components from scorpion venoms,a large number of peptides are identified with an aim of combating a myriad of emerging global health problems.Moreover,some scorpion venom-derived peptides have been established as potential scaffolds helpful for drug development.In this review,we summarize the promising scorpion venoms-derived peptides as drug candidates.Accordingly,we highlight the data and knowledge needed for continuous characterization and development of additional natural peptides from scorpion venoms,as potential drugs that can treat related diseases.

Global Insight into N-Glycome and N-Glycoproteome of Three Most Abundant Snake Venoms in Asia

Snake venom is a complex cocktail including a variety of biological active proteins and proteinaceous components, which have considerable medical and pharmacological importance. N-Glycosylation is widely impli- cated as a common modification in numerous venom proteins and impacts the in vivo venomic functions. However, systematic survey of N-glycome and N-glycoproteome on snake venoms has not been undertaken. In this study, em- ploying combination of N-glycomics and N-glycoproteomics strategies, we explored the N-glycosylation including both N-glycoproteins and N-glyco-chains in three venoms from Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith, respectively, which are amongst the most abundant venomous snakes in Asia. As a result, numbers of N-glycoproteins and N-glycans were identified. However, the overlaps of N-glycoproteins and N-glycans of the three venoms were small. Thus, the exploration results of N-glycome and N-glycoproteome indicate that N-glycosylation increases the complexity and variety of the three venoms. Our research provided some new horizons for the comprehensive understanding of venoms variation, which is helpful for the basic venom re- search as well as the management of snake envenomation.

Risk factors for hemocoagulase-associated hypofibrinogenemia in patients with gastrointestinal bleeding

BACKGROUND With the widespread use of hemocoagulase in patients with gastrointestinal bleeding,clinicians have become increasingly concerned about coagulation dis-orders associated with this medication.Risk factors for hypofibrinogenemia asso-ciated with hemocoagulase are poorly understood.AIM To determine risk factors for hemocoagulase-associated hypofibrinogenemia in patients with gastrointestinal bleeding.METHODS We performed a retrospective analysis of the medical documentation of hospit-alized patients treated with hemocoagulase for gastrointestinal bleeding.Hypofib-rinogenemia was defined as a decrease in plasma fibrinogen concentration to less than 2.0 g/L.The included patients were divided into two groups:acquired hypofibrinogenemia group and non-hypofibrinogenemia group.We used logistic regression analysis to identify potential risk factors and established risk assess-RESULTS There were 36 patients in the acquired hypofibrinogenemia group and 73 patients in the non-hypofibrinogenemia group.The hypofibrinogenemia group showed higher rates of intensive care unit admissions(P=0.021),more female patients(P=0.005),higher in-hospital mortality(P=0.027),larger hemocoagulase doses(P=0.026),more Packed Red Cells transfusions(P=0.024),and lower baseline fibrinogen levels(P<0.000).Binary logistic regression was employed to examine the risk factors associated with acquired hypofibrinogenemia.The analysis revealed that baseline fibrinogen[odds ratio(OR)0.252,95%CI:0.137-0.464,P<0.000],total hemocoagulase doses(OR 1.074,95%CI:1.015-1.137,P=0.014),and female gender(OR 2.856,95%CI:1.015–8.037,P=0.047)were statist-ically significant risk factors.CONCLUSION Higher doses of total hemocoagulase,female gender,and a lower baseline fibrinogen level were risk factors for hemocoagulase-associated hypofibrinogenemia in patients with gastrointestinal bleeding.

STRUCTURE-FUNCTION FEATURES AND EFFECTS ON BLOOD COAGULATION OF SNAKE VENOM SERINE PROTEASES*

Snake venoms,especially those from the two subfamilies,Crotalinae and Viperinae,contained a lot of serine proteases. They were responsible for the hemorrhage,shock,or disorder of blood coagulation after envenomation. They acted,by activating,inactivating,or other converting effects,on almost all the components of hemostatic and fibrinolytic systems. Their sequences were homologous to trypsin-kallikrein serine proteases. Variation of primary sequences out of active center results in the difference of substrate specificities and the further difference of biological and pharmacological activities. Because of their common and unique properties compared to their physiological corresponding factors,snake venom proteases are proved to be an excellent model for the study of protease substrate discriminating mechanism. Furthermore,they have found an important position both in basic research and application of hemostasis and thrombosis in clinic.

Purification and Characterization of Cytotoxins from Agkistrodon acutus Venom and Their Anticancer Activity

Aim To investigate the anticancer activity of two new cytotoxins from thevenom of Agkistrodon acutus. Methods The venom was isolated by FPLC column chromatography consistingof DEAE Sepharose FF and Source 30S. The cytotoxic activity on tumor cells was detected by MITmethod. Purity and molecular weight were determined by SDS-PAGE (silver staining). Their stabilitiesto temperature and pH were also detected. Results Two pure cytotoxins named ACTX-6 and ACTX-8 wereobtained. Their molecular weights are 98 kDa and 27 kDa, respectively. ACTX-6 consists of twosubunits bonded together by disulfide bonds. Conclusion ACTX-6 and ATCX-8 have highest inhibitoryactivity on lung cancer cell A549. ACTX-6 is stable to heat while ACTX-8 not. ACTX-6 is stablebetween pH 7-9 and ACTX-8 between pH 6 - 9.

Isolation and characterization of Hainantoxin-II, a new neurotoxic peptide from the Chinese bird spider (Haplopelma hainanum)

Hainantoxin-II （HnTx-II）, a novel neurotoxin, was isolated from the venom of the Chinese bird spider （Haplopelma hainanum） by cation exchange chromatography and reverse-phase HPLC. The toxin was a single chain polypeptide with calculated molecular weight of 4 253.135 obtained by mass spectrometry. The complete amino acid sequence of HnTx-II was determined by Edman degradation and found to contain 37 residues with three disulfide bonds. Results showed HnTx-II can reversibly paralyze cockroaches for several hours after intra-abdominal injection with ED50 of 16 μg/g and kill the insects immediately at a dose of 60 μg/g. It was also shown to kill mice at a LD50 value of 1.41μg/g after intracerebroventricular injection. Hainantoxin-II shares 91% sequence homology with Huwentoxin-II （HwTx-II）, an insecticidal peptide from another bird spider （Haplopelma schmidti） with a unique scaffold. While HnTx-II and HwTx-II both exhibit toxic activities in insects and mammals, HnTx-II shows higher insecticidal activity and lower lethiferous activity of mammals than HwTx-II. These results help clarify structural-functional relationships of the polypeptide toxin.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Effects of Batroxobin on Spatial Learning and Memory Disorder of Rats with Temporal Ischemia and the Expression of HSP32 and HSP70

The effect of Batroxobin on spatial memory disorder of left temporal ischemic rats and the expression of HSP32 and HSP70 were investigated with Morri`s water maze and immunohistochemistry methods. The results showed that the mean reaction time and distance of temporal ischemic rats in searching a goal were significantly longer than those of the sham-operated rats and at the same time HSP32 and HSP70 expression of left temporal ischemic region in rats was significantly increased as compared with the sham-operated rats. However, the mean reaction time and distance of the Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of HSP32 and HSP70 immune reactive cells of Batroxobin-treated rats was also less than that of the ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats; and the down-regulation of the expression of HSP32 and HSP70 is probably related to the attenuation of ischemic injury.

Effects of cosmetics containing purified honeybee(Apis mellifera L.) venom on acne vulgaris

Acne vulgaris is a chronic dermatologic problem with multiple factors involved in its pathogenesis. Alternative solutions to acne treatment were instigated by antibiotic resistance despite of its extensive use. Purified bee venom （PBV） has been proposed as a promising candidate for that purpose. The present study was designed to confirm the antibacterial effect of PBV and access the efficacy of cosmetics containing PBV in subjects with acne vulgaris. METHODS： The skin bacterium Propionibacterium acnes was incubated with PBV at various concentrations and bacterial growth was evaluated using the colony forming unit （CFU） assay. The mechanism of PBV employed in killing P. acnes was examined by scanning electron microscopy （SEM） and transmission electron microscopy （TEM）. In addition, a total of 12 subjects were randomized in a double-blind, controlled trial to receive either cosmetics containing PBV or cosmetics without PBV for two weeks. Evaluations included lesion counts and skin microorganism. RESULTS： PBV exhibited antimicrobial activity in a concentration-dependent manner, reducing the number of P. acnes CFU by approximately 6 logs at a concentration of 0.5 mg. When PBV concentration was higher than 1.0 mg, no P. acnes colonies were spotted on an agar. TEM and SEM of untreated P. acnes illustrated the normal pleomorphic structure, whereas the PBV- treated bacterium lost the integrity of surface architecture. Significant difference （P=0.027） in the grading levels based on numbers of lesion counts for inflammatory and noninflammatory was observed in favour of the PBV group compared with the control group. In terms of average decrement of skin microorganism, subjects receiving cosmetics containing PBV experienced a significant 57.5% decrease of adenosine triphosphate levels, whereas participants receiving cosmetics without PBV experienced a nonsignificant decrease of 4.7%. CONCLUSION： These results show that the in vitro actions of antimicrobial activity of PBV were translated in vivo. Cosmetics containing PBV provided a certain degree of efficacy in terms of lesion counts and skin microorganism concentration compared with cosmetics without PBV in subjects with acne vulgaris. PBV may be a good candidate compound for developing therapeutic drua for the treatment of acne vulaaris.

Primary Study of Egg Yolk Antibody for Detection of King Cobra Venom Antigens

Aim To study whether antivenom from laying hens can be used for the detection of venom antigens, Methods Chickens （white Leghorn） were immunized with detoxicated king cobra venom by formaldehyde and egg yolk immunoglobulin （IgY） isolated from yolk; IgY was labelled with the horseradish peroxidase （HRP）. Experimental condition and parameters were determined by chessboard test. The specificity, sensitivity, precision, and stability of this method were assayed in the experiment. Results This method could detect as low as 32 μg· L^-1 of the king cobra antigens. A good linear relation was found within 32 ～ 750 μg· L^-1 of king cobra venom concentrations （ r = 0. 963）. There was no cross reactivity for the reagents with Agkistrodon acutus Guenther venom or Vipera russelli siamensis Smith venom;slight cross reactivity .with Bungarus multicinctus Blyth venom or Bungarus fasciatus Chmeider venom; and notable cross reactivity with cobra venom. The average intra-assay relative standard deviation （RSD） was 1% - 3%, and the inter-assay RSD was less than 8%. The reagents （including IgY and HRP-IgY） were stable; no differences （P 〉 0.05） were observed for the detection of venom antigens when the reagents were stored at 37 ℃ up to 6 d. Conclusion IgY is a good reagent for diagnosis of snakebite after eliminating the genus cross reactivity.

The effects of scorpion(Buthus martensi Karsch)venom on rat left ventricular function

In order to investigate the cardiovascular effects of the scorpion(Buthus martensiKarsch)venom(BmKv),the left ventricle of the rats was catheterized via the right carotidartery.The LVP,LVEDP,+dp/dt max,Vmax,HR and BP were observed.The results showedthat intravenous injection of the BmKv(60μg/kg),in comparison with the control,elicited obvi-ous hypertension and increase of cardiac contractility,both of which lasted for 1h,while theheart rate had no significant change rand that pretreating the rats with alpha-adrenergic blocker,phentolamine,antagonized the hypertensive effects,but did not antagonize the increase of cardiaccontractility.Pretreatment with beta-adrenergic blocker,propranolol,has no influence on the ef-fects of the venom.It is suggested that the hypertensive effects are due to the activation of al-pha-adrenergic receptor,whereas the increase of cardiac contractility may not be resulted fromthe activation of beta-adrenergic receptor.The BmKv treated with dithiothreitol before injectionhad no cardiovascular effects,indicating that the intact disulfide bridges play a decisive role inthe cardiovascular effects of the BmKv.

Essential Oils from Mentha piperita,Cymbopogon citratus,Rosmarinus officinalis,Peumus boldus and Foeniculum vulgare:Inhibition of Phospholipase A_(2) and Cytotoxicity to Human Erythrocytes

The essential oils from Mentha piperita, Cymbopogon citratus, Rosmarinus officinalis, Peumus boldus and Foeniculum vulgare were extracted by hydrodistillation and characterized and quantified by GC-MS and GC-DIC. The oils induced hemolysis with all the doses evaluated (0.6 to 1.8 μL), and the diameters of the halos varied between 9 and 15 mm. Pre-incubation of P. boldus oil with Bothrops jararacussu venom resulted in potentiation of venom-induced hemolysis (30%) (proteases and phospholipases A2). The essential oil from M. piperita (0.6 μL) inhibited venom-induced hemolysis by 45%, whereas 0.6 μL of R. officinalis oil increased the hemolysis by 20%. For the essential oil from F. vulgare, 100% inhibition of activity (0.6 and 1.2 μL) was observed. The application of C. citratus oil induced hemolysis with all the volumes evaluated. Phospholipase activity induced by the venom was only inhibited (10%) with the 0.6 μL volume of R. officinalis oil. The oils from M. piperita and F. vulgare (1.8 μL) and C. citratus oil (0.6 μL) potentiated the phospholipase activity. The results highlight the need for a broad characterization and regulation of the use of natural products, because they can have therapeutic or toxic actions.

Anti-trypanosomal Activity of Bufonidae(Toad)Venom Crude Extract on Trypanosoma brucei brucei in Swiss Mice

Trypanosomiasis afflicts about 6~7 million people globally and to a large extent impedes livestock production in Africa.Naturally,trypanosomal parasites undergo genetic mutation and have developed resistance over a wide range of therapies.The utilization of animals and plants products has presented therapeutic potential for identifying novel anti-trypanosomal drugs.This study evaluated toad venom for anti-trypanosomal potency in-vivo in Swiss mice.Toads were collected from July to August 2019.The acute oral toxicity and biochemical characterization of the toad venom were determined.The experimental mice were administered various doses(130 mg/kg,173 mg/kg and 217 mg/kg)of the toad venom crude extract and 0.75 mg/mL of Diamizan Plus standard drug for the treatment of trypanosomiasis,once daily for 3 days.The in-vivo anti-trypanosomal activity was evaluated by a curative test,after infecting the mice with Trypanosoma brucei brucei.The pre-patent period was 72 hours before treatment commenced.The overall results showed that trypanosomal load was highest in the control group while the group treated with Diamizan drug had the least trypanosomal load.As such,the mean trypanosomal load in relation to treatments showed a very high significant difference(P<0.05).Also,the mean trypanosomal load in Swiss mice in relation to the highest dosage of toad venom versus Diamizan drug showed a very high significant difference(P<0.05).The mean change in relation to the haematological parameters across treatments groups varied significantly(P<0.05)with the exception of Hb which showed no significant difference(P>0.05)across treatment groups.The over 50%reduction in the trypanosomal load in the 130 mg/kg group in comparison with the control group brings to bare the anti-trypanosomal potency of the toad venom.The anti-trypanosomal activity demonstrated by the toad venom has provided basis for development of new therapeutic agents from different toad species.The study recommends further studies(both in-vivo and in-vitro)followed by the characterization of the active compounds present in the toad venom responsible for the anti-tyrpanosomal activity observed alongside the management and conservation of these species.

PURIFICATION AND CHARACTERIZATION OF A NEW FIBRINOGENASE FROM THE VENOM OF THE CHINESE HABU SNAKE (Trimeresurus mucrosquamatus)

A new fibrinogenase(EC 3.4.2 I.5)was isolated and purified from the venom of Chinese habu snake(Trimeresurus研ucrosg“ama似s)by DEAE—SephadexA-50,DEAE—Sepharose CL一6B,MonoQ(FPLC)CG.1umn chromatography.It showed a single protein band both in sodium dodeeyl sulfate(SDS)·polyacrylami-de geI electrophoresis and alkaline polyacrylamide gel electrophoresis.The molecular weight was estimated tobe 26000 by SDS·p01yacrylam|de gel electrophoresis.The isoeleetric point was found to be pH 4.7.Itwas a glycoprotein containing 6.4‘%carbohydrate with o.3%neutral sugar,1.2%sialic acid,4.9%he.xosamine.It was composed of about 1 78 amino acid residues and rich in glycine and aspartic acid.Thefibrinogenase of the venom of T.munro$quclmatu$TWV№was heat stable but labile to acid.Its extinctioncoefficient(1mg/m1)at 280rim was 1.558.Purified TMVFg had strong arginine esterase activity·the Kmto benzoylarginine ethylester(BAEE)was 1.4×1 0一M.The enzyme activity could be inhibited by pheny.1mefha口esulfonyfluoride(PMSF),but was not affected by ethylenediamine tetraacetlc acid(EDTA).TMVFghad fibrinogenolytie activityl electrophoresis of fibrinogen degraded with TMVFg revealed the rapiddisappearance of the口(alpba)and B(beta)‘chains and the appearance I】f lower molecular weight frag.merits.TMVFg did not cause fibrinogen solution clotting,nor coagulating plasma and showed n^ither hemorr-hagic activity nor proteolytic activity toward casein.TMVFg had activati^lg fibrinolytic activity

Therapeutic potential of snake venom in cancer therapy:current perspectives

Many active secretions produced by animals have been employed in the development of new drugs to treat diseases such as hypertension and cancer.Snake venom toxins coutributed significantly to the treatment of many medical conditions.There are many published studies describing and elucidating the anti-cancer potential of snake venom.Cancer therapy is one of the main areas for the use of protein peptides and enzymes originating from animals of different species.Some of these proteins or peptides and enzymes from snake venom when isolated and evaluated may bind specifically to cancer cell membranes,affecting the migration and proliferation of these cells.Some of substances found in the snake venom present a great potential as anti-tumor agent.In this review,we presented the main results of recent years of research involving the active compounds of snake venom that have anticancer activity.

Therapeutic Potential of Naja Naja Atra Venom in A Rat Model of Diabetic Nephropathy

Objective To study the protective effects of naja naja atra venom （NNAV） in a rat model of diabetic nephropathy （DN）. Methods The rat diabetes model was induced by intraperitoneal injection of streptozotocin （STZ）. Thirty-two model rats were randomly divided into one DN group （n=8） and three treatment groups （n=8 each） that received NNAV at doses of 30, 90, or 270 I^g/（ks.day） via oral gavage, another eight rats as normal controls. After 12 weeks, all rats were sacrificed and the changes in serum and urine biological index levels were determined by colorimetric assay. Microalbumin （mALB）, N-acetyl-13- glucosaminidase （NAG） and cystatin C （CysC） concentrations were measured by ELISA. Renal tissues were sliced for pathological and immunohistochemical observations. Results Comparied with the DN group, serum glucose was decreased by 31.04%, total cholesterol 21.96%, triglyceride 23.78%, serum creatinine 19.83%, blood urea nitrogen 31.28%, urinary protein excretion 45.42%, mALB 10.42%, NAG 20.65%, CysC 19.57%, whereas albumin increased by 5.55%, high-density lipoprotein-cholesterol 59.09%, creatinine clearance 19.05% in the treatment group by NNAV administration at dose of 90 μg/（kg-day）. NNAV also reduced the levels of malondialdehyde in serum （22.56%） and kidney tissue （9.79%）, and increased superoxide dismutase concentration in serum （15%） and decreased it in renal tissue （8.85%）. In addition, under light microscopy kidney structure was improved and glomerular hypertrophy decreased by 8.29%. As shown by immunohistochemistry, NNAV inhibited transforming growth factorl by 6.70% and nuclear actor-KB by 5.15%.

Systematics and Species Validity of the Dabieshan Pit Viper Protobothrops dabieshanensis Huang et al. 2012: Evidence from a Mitochondrial Gene Sequence Analysis

Aimed to evaluate the phylogenetic position of the recently described Protobothrops dabieshanensis Huang et al. （2012）, phylogenic relationships of 12 species within Protobothrops based on four mtDNA gene fragments （12S RNA, 16S RNA, ND4 and Cyt b） were reconstructed in our study. The result indicates a clade composed ofP dabiesha- nensis, P. jerdonii and P xiangchengsis with strong support. The genetic distance among P dabieshanensis, P jerdonii and P xiangchengsis was much lower than other congeners. Based on the data from the phylogenetic analysis and pre- viously described morphological differences, we conclude that P dabieshanensis is a valid species with close affinities to P jerdonii and P xiangchengsis.