Malabarase,a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152–341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.     

Cost of venom regeneration in Parabuthus transvaalicus (Arachnida: Buthidae)

Scorpion venom has many components, but is mainly made up of water, salts, small molecules, peptides, and proteins. One can reasonably assume that the production and storage of this complex secretion is an expensive metabolic investment. However, to date, no study has addressed the costs associated with the regeneration of venom by scorpions. Using a closed-system respirometer, we examined the difference in oxygen consumption between milked and unmilked scorpions to determine the metabolic costs associated with the first 72 h of subsequent venom synthesis. During this time period, milked scorpions had a significantly higher (39%) metabolic rate than unmilked scorpions. The regenerated venom from a second milking had significantly lower (74%) protein concentration, suggesting that venom regeneration was incomplete after 72 h. The protein content in the regenerated venom was not correlated with oxygen consumption. The significant increase in oxygen consumption after milking supports existing hypotheses about the metabolic cost associated with venom regeneration and provides further insight on why scorpions appear to be judicious in their stinger use.     

Construction and characterization of a cDNA library from shark regenerated hepatic tissue

Sharks are a type of fish with a full cartilaginous skeleton and have big livers. To better understand liver regeneration in sharks and screening for the important genes participated in disease-defense, in this study, a cDNA library of regenerated liver tissues of shark, Chiloscyllium plagiosum, was constructed. A total of 2103 expressed sequence tags (ESTs), which represents 997 unique genes, were sequenced. Among these genes, 434 (43.53%) of them showed significant similarity (E-values < 10^?5) to the sequences in NCBI Nt database, 685 (68.71%) of these unique genes showed significant similarity (E-values < 10^?5) to the sequences in NCBI Nr database, and 662 (66.40%) of these unique genes showed significant similarity (E-values < 10^?5) to the Swiss-Prot database. Preliminary analysis of unique genes according to COG database showed that unigenes were further grouped into 21 functional categories including inorganic ion transport and metabolism, energy production and conversion, posttranslational modification, protein turnover and chaperones, general function prediction only, translation, and ribosomal structure and biogenesis. Several possible candidate genes involved in liver regeneration were selected to analyze their expression with relative quantification real-time PCR. This study may contribute to our better understanding of the molecular mechanism of regeneration in shark liver. Furthermore, the EST cataloguing and profiling of shark will be also benefited to the functional genomic research in this marine species.     

Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus

We previously reported that bumblebee (Bombus ignitus) venom serine protease (Bi-VSP) acts as a prophenoloxidase-activating factor in arthropods and a fibrin(ogen)olytic enzyme in mammals. In the present study, we characterized the enzymatic properties of Bi-VSP purified from B. ignitus venom. The 34-kDa active form of Bi-VSP was purified from the venom of B. ignitus worker bees. Glycoprotein staining showed that approximately 20% of the total molecular mass of Bi-VSP is due to carbohydrate moieties. Bi-VSP had an optimal pH and temperature of pH 9.0 and 40 °C, respectively, and was stable at 50 °C for at least 10 min. Bi-VSP activity decreased abruptly below pH 6.0, indicating that Bi-VSP activity is almost completely inhibited at pH 5.4 of B. ignitus venom. The protease activity of Bi-VSP was strongly inhibited by typical serine protease inhibitors such as phenylmethanesulfonyl fluoride, leupeptin, and soybean trypsin inhibitor.     

The economic effects of an estrus synchronization protocol using prostaglandin in beef heifers

We estimated the effect of estrus synchronization on reproduction, production and economic outcomes in 272 beef heifers randomly allocated to a synchronized Test group or an unsynchronized Control group. The Test group received AI upon estrus detection for 6 days followed by PGF2 treatment of heifers that had not shown estrus by day 6 (PGF/6). In both groups AI was continued for 50 days, followed by a 42-day bull breeding period. Heifers were followed through their second breeding season and until they had weaned their first calves. Synchronization resulted in a reduction in median days to first insemination (8 vs. 11 in the Test and Control groups, respectively, P < 0.01) and median days to calving of calves born to AI (14 vs. 20, P = 0.04). There was no significant difference in pregnancy rate to the AI period (60.0% vs. 51.8%, P = 0.18), final pregnancy rate (82.2% vs. 83.2%, P = 0.87) or pregnancy rate to the subsequent breeding season (96.0% vs. 95.0%, P = 1.00). Although mean calf weaning mass was not significantly different (207.0 kg vs. 201.4 kg, P = 0.32), the total mass of calves weaned in this study was 14,843 kg vs. 13,060 kg and the benefit: cost ratio for synchronization was 2.8. It was therefore concluded that a PGF/6 protocol may affect the total mass of calves weaned by changing days to calving, weaning rate, the ratio of male: female calves born and/or the birth mass of calves.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65 °C over 6 h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.     

Size exclusion chromatography of zein and xanthophylls: Optimization of operating parameters

Ethanol-soluble components of corn (zein and xanthophylls) were separated and purified by size exclusion chromatography. Aqueous ethanol was used as the solvent for the entire process from extraction through chromatography. The effect of operating and design parameters, such as temperature, flow rate, loading mass, loading volume, column height and diameter, on productivity and resolution of the components was studied. Using a one-variable-at-a-time (OVAT) approach with 1 cm × 60 cm columns, optimum conditions were determined to be 40 °C temperature, 0.25 mL/min flow rate, volume loading of 22 mL corn extract, mass loading at 70 g/L of corn extract. All components could be resolved with base line separation with a 240 cm column length. Column diameter did not affect separation, implying linear scalability with constant flow distribution.     

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.     

Batch culture and repeated-batch culture of Cunninghamella bainieri 2A1 for lipid production as a comparative study

This research was performed based on a comparative study on fungal lipid production by a locally isolated strain Cunninghamella bainieri 2A1 in batch culture and repeated-batch culture using a nitrogen-limited medium. Lipid production in the batch culture was conducted to study the effect of different agitation rates on the simultaneous consumption of ammonium tartrate and glucose sources. Lipid production in the repeated-batch culture was studied by considering the effect of harvesting time and harvesting volume of the culture broth on the lipid accumulation. The batch cultivation was carried out in a 500 ml Erlenmeyer flask containing 200 ml of the fresh nitrogen-limited medium. Microbial culture was incubated at 30 °C under different agitation rates of 120, 180 and 250 rpm for 120 h. The repeated-batch culture was performed at three harvesting times of 12, 24 and 48 h using four harvesting cultures of 60%, 70%, 80% and 90%. Experimental results revealed that nitrogen source (ammonium tartrate) was fully utilized by C. bainieri 2A1 within 24 h in all agitation rates tested. It was also observed that a high amount of glucose in culture medium was consumed by C. bainieri 2A1 at 250 rpm agitation speed during the batch fermentation. Similar results showed that the highest lipid concentration of 2.96 g/L was obtained at an agitation rate of 250 rpm at 120 h cultivation time with the maximum lipid productivity of 7.0 × 10⁻² mg/ml/h. On the other hand, experimental results showed that the highest lipid concentration produced in the repeated-batch culture was 3.30 g/L at the first cycle of 48 h harvesting time using 70% harvesting volume, while 0.23 g/L gamma-linolenic acid (GLA) was produced at the last cycle of 48 h harvesting time using 80% harvesting volume.     

Characterisation of neurotoxic polypeptides from <Emphasis Type="Italic">Cyanea capillata</Emphasis> medusae (Scyphozoa)

Cnidarian venoms include neurotoxins, which are able to paralyse prey organisms immediately. Important targets for neurotoxins are voltage-gated ion channels in membranes of excitable cells. By blocking specific receptor sites, neurotoxic components disturb the physiological ion channel functions. Here, we describe the isolation and characterisation of potential neurotoxic polypeptides from the crude tentacle venom of the boreal scyphomedusan Cyanea capillata. Partially purified venom fractions were obtained by size-exclusion and subsequent reversed-phase chromatography. To assess the blocking activity of the venom on voltage-gated sodium channels, we modified a mouse neuroblastoma (MNB) cell assay. Venom fractions containing channel-blocking activity were analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The resulting mass spectra revealed a cluster of singly charged peptides within a mass range from 3,900 to 7,000 Da. A group of three potentially neurotoxic peptides with molecular masses of 3983.4, 5795.4 and 6962.1 Da could be tracked throughout the purification process. This investigation of the crude venom is part of a multidimensional assay-guided approach for the isolation and structural characterisation of toxic polypeptides in northern Scyphozoa.     

Possible recovery from an acute envenomation in male rats with LD50 of Echis coloratus crude venom: I-A seven days hematological follow-up study

The effect of an acute LD₅₀ dose of Echis coloratus crude venom in male albino rats was tested on blood parameters: white blood cells (WBCs), red blood cells (RBCs), platelets count, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), also serum glucose, total protein, triglycerides with alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) enzyme activities. The effect of the LD₅₀ dose was monitored over a period of seven days, with time intervals of 1, 3, 6, 12, 24, 72 h. All of the tested parameters show fluctuations with time and with tendency to regain normal control level after 12 h. At 12–24 h it seems to be crucial for the process of physiological recovery, in spite of the irreversible damage and tissue distraction. The process of physiological adaptation and recovery from the lethal destructive venom effect seems to stabilize after one week, leaving the animal alive with several biochemical altered metabolisms and disturbed physiological profile.     

Comprehensive gas chromatography with Time of Flight MS and large volume introduction for the detection of fluoride-induced regenerated nerve agent in biological samples

Recently, several methods have been developed to verify exposure to nerve agents. Most of these methods, such as the fluoride reactivation technique and the analysis of inhibited phosphonylated butyrylcholinesterase (BuChE), are based on mass spectrometry. The high specificity of the mass spectrometer might also imply a disadvantage, because the acquisition mass, i.e. the identity of the analyte must be known beforehand in order to direct the MS analysis in the most sensitive mode. In real cases, the identity of the nerve agent is not always known beforehand and the mass spectrometer should be operated in a scanning mode, with the consequence that sensitivity of the method will be lower. Comprehensive GC, or GC × GC, is a technique which offers enhanced separation. The implied larger selectivity of the GC separation allows mass spectrometry to be conducted in a less specific, scanning, mode. By the use of this configuration, the identity of the nerve agent does not have to be known beforehand but can be traced. In order to be able to detect lower concentrations and assess lower exposure levels, a large volume injection technique was developed allowing sample sizes up to 100 μL. The technique was tested with plasma samples that had been inhibited with various nerve agents. Subsequently, the cholinesterase-bound nerve agent was regenerated by the fluoride reactivation technique. Using the newly developed comprehensive GC–MS method it was possible to detect nerve agent at an exposure level of 1% BuChE inhibition, which is approximately 70 pg nerve agent/mL. These low exposure levels cannot be verified with a cholinesterase (ChE) activity assay. Moreover, the identity of the regenerated nerve agent was verified by the mass spectrum that was generated by the TOF mass spectrometer. This paper presents a technique able to deliver full-scan data on the analysis of nerve agents in biomedical samples at relevant exposure levels (1% BuChE inhibition). This full-scan data meets for a large part the forensic requirements that are in place for the analysis of biomedical samples in the context of alleged use of Chemical Warfare Agents.     

Puma spatial ecology in open habitats with aggregate prey

Solitary felids are commonly associated with structurally complex habitats, where their foraging success is attributed to stealth and remaining undetected by competitive scavengers. Research in North America suggests that pumas (Puma concolor), a wide-ranging species found throughout the Americas, conform to the general characteristics of solitary felids and avoid open grasslands with aggregating prey. Researchers hypothesize that pumas are limited to structurally complex habitats in North America because of pressures from other large, terrestrial competitors. We explored the spatial ecology of pumas in open habitat with aggregating prey in Chilean Patagonia, where pumas lack large, terrestrial competitors. We tracked 11 pumas over 30 months (intensive location data for 9 pumas with GPS collars for 9.33 ± 5.66 months each) in an area where mixed steppe grasslands composed 53% of the study area and carried 98% of available prey biomass, to track resource use relative to availability, assess daily movements, quantify home ranges and calculate their density. As determined by location data and kill sites, Patagonia pumas were primarily associated with open habitats with high prey biomass, but at finer scales, preferentially selected for habitat with complex structure. On average, pumas traveled 13.42 ± 2.50 km per day. Estimated 95% fixed kernel home ranges averaged 98 ± 31.8 km² for females and 211 ± 138.8 km² for males, with high spatial overlap within and between the sexes. In a multivariate analysis, available prey biomass was the strongest predictor of variation in the size of an individual puma's home range. Finally, we determined a total puma density of 3.44 pumas/100 km², a significantly smaller estimate than previously reported for Patagonia, but similar to densities reported for North America.     

Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn.—An endangered plant

Leptadenia reticulata (Retz.) Wight. & Arn. is an important medicinal plant, belongs to the family Asclepiadaceae. This plant is known for its medicinal uses since 4500 BC. Presently this is an endangered species (Arya et al., 2003). Six shoots (2–4 cm long) per node differentiated on MS medium + 5.0 mg/l of BAP + additives. Incorporation of additives in the culture medium promoted growth of cultures. The shoots differentiated per explant were repeatedly transferred on to fresh MS + 1.0 mg/l of BAP + 0.1 mg/l of NAA and additives. The regenerated shoots were subcultured for further multiplication on MS + 1.0 mg/l BAP + 0.5 mg/l Kin + 2-iP (0.5 mg/l) and 0.1 mg/l of NAA + additives regularly after an interval of 3 weeks. Addition of ammonium sulphate in the medium resulted in increase in shoot number and promoted elongation also growth of cultures was sustained even if subculturing was delayed (26 ± 2 days). Success was also achieved in defining protocol for in vitro regeneration of shoots from petiole derived callus. Shoots regenerated in vitro by both processes were rooted in vitro on 1/4 strength of MS medium + 3.0 mg/l of IBA after 15–20 days. Cent percent of the shoots rooted ex vitro, if the in vitro regenerated shoots were treated with 200 mg/l of IBA. The in vitro–ex vitro rooted plantlets were hardened under different regimes of temperature and humidity in a greenhouse. The hardened plantlets were transferred to soil in polybags. More than 95% plants survived in field conditions. Total dry biomass harvested per year was 2800 kg/acre.     

Non-disulfide-bridged peptides from Tityus serrulatus venom: Evidence for proline-free ACE-inhibitors

The present study purifies two T. serrulatus non-disulfide-bridged peptides (NDBPs), named venom peptides 7.2 (RLRSKG) and 8 (KIWRS) and details their synthesis and biological activity, comparing to the synthetic venom peptide 7.1 (RLRSKGKK), previously identified. The synthetic replicate peptides were subjected to a range of biological assays: hemolytic, antifungal, antiviral, electrophysiological, immunological and angiotensin-converting enzyme (ACE) inhibition activities. All venom peptides neither showed to be cytolytic nor demonstrated significant antifungal or antiviral activities. Interestingly, peptides were able to modulate macrophages’ responses, increasing IL-6 production. The three venom peptides also demonstrated potential to inhibit ACE in the following order: 7.2 > 7.1 > 8. The ACE inhibition activity was unexpected, since peptides that display this function are usually proline-rich peptides. In attempt to understand the origin of such small peptides, we discovered that the isolated peptides 7.2 and 8 are fragments of the same molecule, named Pape peptide precursor. Furthermore, the study discusses that Pape fragments could be originated from a post-splitting mechanism resulting from metalloserrulases and other proteinases cleavage, which can be seen as a clever mechanism used by the scorpion to enlarge its repertoire of venom components. Scorpion venom remains as an interesting source of bioactive proteins and this study advances our knowledge about three NDBPs and their biological activities.     

Gene expression of apoptosis-related genes,stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.     

Reproductive aspects of American minks (Neovison vison) in central Spain: Testing the effects of prey availability

Some reproductive components of fitness (breeding times, litter size and the proportion of breeding females per year) of two feral populations of American minks in central Spain were investigated by means of direct observation from 2006 to 2010. The effects of prey availability were explored. Mating season was from February to March. Parturition dates did not differ between the two sites and pooled births took place between April (33.3%) and July (2.8%), peaking in May (40.3%). Mean litter size (the number of small cubs following their mother) was 3.43 ± 1.01 cubs (±SD; n = 30) with statistical significant differences between the two study sites. The strong seasonality in births and differences in litter size were related to prey availability (i.e. these are food-limited life-history traits), but in a complex non-linear fashion. A minimum value of prey abundance is necessary to breed. While litter size rises with prey abundance, there is a point where increasing prey did not result in an increase in litter size. Up to 75% of the females in the breeding pool reared cubs each year in both areas, which represents a somewhat high pool of breeding females. Delayed implantation is shorter than a month or does not exist in these populations. Such a short time-span seems to emerge because longer delays would have an important fitness cost to females. The breeding calendar and the litter size were similar to that reported previously from other areas.