Sephadex Gel Filtration Research Articles

A novel low temperature chitinase from the marine fungus Plectosphaerella sp. strain MF-1

The marine fungus Plectosphaerella sp. strain MF-1 was isolated from sea shells and found to produce a chitinase potentially active at low temperature. The fungal strain was characterized by morphological and molecular features. Chitinase production by Plectosphaerella MF-1 was detected by inoculating the fungus into M9 medium containing 0.5% colloidal chitin at 10°C. Crude chitinase production in culture filtrates reached a maximum 14 days after inoculation. Crude chitinase was purified by ammonium sulfate fractionation, cellulose DEAE anion exchange, and sephadex gel filtration chromatography. Purified marine fungal chitinase had activity at 37°C, the difference in chitinase activities at 10°C and 37°C was less than 0.01 U ml -1 indicating chitinase was active at low temperature. The optimal pH for the low temperature active chitinase was 3–4. The K m was 0.03 m m and V max was 0.095, using p -nitrophenyl N -acetyl-β- d -glucosaminide as a substrate. Among the metal ions tested for inhibitory activity, Ag + , Hg 2+ , and Pb 2+ strongly inhibited enzyme activity, whereas Mg 2+ and Fe 2+ had minimal inhibition. The molecular mass of purified chitinase was determined as 67 kDa by SDS-PAGE. The N-terminal amino acid sequence was determined to be “DNISQTGEHARYXPMVWFIKL”.

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 degrees C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203.

Two novel antimicrobial peptides from centipede venoms

Centipede venoms are complex mixtures of biochemically and pharmacologically active components such as peptides and proteins. Very few are known about their pharmacological actions. The present work reports the structural and functional characterization of two antimicrobial peptides (scolopin 1 and -2) identified from centipede venoms of Scolopendra subspinipes mutilans by Sephadex gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). The amino acid sequences of scolopin 1 and -2 were FLPKMSTKLRVPYRRGTKDYH and GILKKFMLHRGTKVYKMRTLSKRSH determined by Edman degradation and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Both scolopin 1 and -2 showed strong antimicrobial activities against tested microorganisms including Gram-positive/negative bacteria and fungi. They also showed moderate hemolytic activity against both human and rabbit red cells. This is the first report of antimicrobial peptides from centipedes.

Evaluation of antagonistic activities of Bacillus subtilis and Bacillus licheniformis against wood-staining fungi: In vitro and in vivo experiments

The antifungal activity of bacterial strains Bacillus subtilis EF 617317 and B. licheniformis EF 617325 was demonstrated against sapstaining fungal cultures Ophiostoma flexuosum, O. tetropii, O. polonicum, and O. ips in both in vitro and in vivo conditions. The crude active supernatant fractions of 7 days old B. subtilis and B. licheniformis cultures inhibited the growth of sapstaining fungi in laboratory experiments. Thermostability and pH stability of crude supernatants were determined by series of experiments. FT-IR analysis was performed to confirm the surface structural groups of lipoproteins present in the crude active supernatant. Partial purification of lipopeptides present in the crude supernatant was done by using Cellulose anion exchange chromatography and followed by Sephadex gel filtration chromatography. Partially purified compounds significantly inhibited the sapstaining fungal growth by in vitro analysis. The lipopeptides responsible for antifungal activity were identified by electrospray ionization mass spectrometry after partial purification by ion exchange and gel filtration chromatography. Four major ion peaks were identified as m/z 1023, 1038, 1060, and 1081 in B. licheniformis and 3 major ion peaks were identified as m/z 1036, 1058, and 1090 in B. subtilis. In conclusion, the partially purified lipopeptides may belong to surfactin and iturin family. In vivo analysis for antifungal activity of lipopeptides on wood was conducted in laboratory. In addition, the potential of extracts for fungal inhibition on surface and internal part of wood samples were analyzed by scanning electron microscopy.

Preparation and Antioxidative Properties of a Rapeseed (Brassica napus) Protein Hydrolysate and Three Peptide Fractions

This study investigated the possibility of converting the insoluble rapeseed meal protein into functionally active ingredients for food applications. The rapeseed ( Brassica napus ) meal protein isolates were first digested by Alcalase and Flavourzyme, and the resultant rapeseed crude hydrolysate (RSCH) exhibited a dose-dependent reducing antioxidant power and hydroxyl radical scavenging ability. RSCH could also inhibit the malonyldialdehyde (MDA) generation by 50% in blood serum at 150 mg/mL. RSCH was further separated into three fractions (RSP1, RSP2, and RSP3) by Sephadex gel filtration according to their different molecular weights. The amino acid compositions and antioxidant potentials were assessed for RSP1-3 fractions. All three fractions showed inhibiting effects on superoxide anion generation to various extents. They could also inhibit the autohemolysis of rat red blood cells and MDA formation in rat liver tissue homogenate. The results suggested that rapeseed peptide hydrolysate may be useful as a human food addition as a source of bioactive peptides with antioxidant properties.

Enrichment of X-spermatozoa and in vitro penetration through cervical mucus.

Using the Sephadex Gel-filtration method (Steeno - 1975) we carried out a review of the data on the possibility of enriching X-spermatozoa. The testing procedure followed consisted of determining Y-chromatin positive spermatozoa after staining with quinacrine-mustard. We were able to confirm the data recorded by Steeno, according to which an enrichment of about 80-90% can be achieved after penetration through the Sephadex Gel filter. In addition to these investigations we also carried out tests with the enriched X-spermatozoa material to determine in vitro penetration through cervical mucus. No decline in the progressive motility of the spermatozoa was observed. After the Gel-filtration sufficient numbers of these were still able to cover penetration distances of 60 mm/hour. The enrichment effect of the X-spermatozoa declined at the beginning of in vitro penetration, but there was no further change in the subsequent course of penetration.

Separation of X- and Y-bearing human spermatozoa with the sephadex gel-filtration method.

By means of the Sephadex gel filtration method we were able to separate a fraction rich in X-bearing human spermatozoa, in contrast with the method of Ericsson et al. (1973), which allows to obtain a fraction rich in Y-bearing sperm. The method, which is simple and not time-consuming, may allow control of sex in cases of expressed sex desire in off-spring.

The separation of X-and Y-spermatozoa with regard to the possible clinical application by means of artificial insemination.

With the sephadex gel-filtration method according to Steeno and Adimoelja (1974), X-spermatozoa have been successfully separated from the Y-spermatozoa in semen samples from 81 donors with the possibility of practical use with the aid of artificial insemination to promote pregnancy. A better quality of spermatozoa is obtained after this method of filtration, which will support these spermatozoa in having the potency of fertilization. There is indeed a decrease in concentration of spermatozoa after filtration, but the samples still fulfil the minimal concentration required in artificial insemination.

Withania somnifera (Ashwagandha): a Novel Source of L‐asparaginase

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 degrees C. The Km value for the enzyme is 6.1 x 10(-2) mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant.

Isolation of 5-Hydroxytryptamine from the Venom of the Indian Red Scorpion (Mesobuthus tamulus)

The present study was undertaken to isolate 5-hydroxytryptamine (5-HT) from crude Indian red scorpion (Mesobuthus tamulus; MBT) venom as it has not been reported yet. Therefore, with the help of bioassay (using gastric fundus contractions) and fluorimetric assay methods, the 5-HT content of crude MBT venom (CV) was determined. The concentration of 5-HT in CV by either of the methods was different (117 times). Further, a 5-HT antagonist methysergide partially blocked the CV-induced gastric fundus contractions. Hence, for isolation and determination of 5-HT in crude MBT venom, CV was subjected to Sephadex gel (G 75-125) filtration chromatography. The eluates were screened for op- tical density (OD) at 280 nm, lethality and gastric fundus contractions. Two peaks of enhanced gastric fundus contractions were observed between 26-70 ml and 90 -110 ml of eluate volumes. As the first peak (between 26-70 ml) eluates exhib- ited high OD and lethality, further analysis was not undertaken. The second peak (between 90-110 ml) eluates exhibited very low OD and were non-lethal. On bioassay, the 5-HT content of pooled eluates of second peak was 0.0398 ± 0.008 � g/ml and by fluorimetric assay it was 0.0396 ± 0.007 � g/ml. The 5-HT concentration of crude MBT venom per unit mass was 0.0175 ± 0.0035 �g/mg by bioassay and 0.0174 ± 0.0031 �g/mg by fluorimetric assay. Further, the gastric fundus contractions elicited by second peak eluates were blocked by methysergide (1.0 �M). Thus, the second peak eluates of crude MBT venom contain 5-HT and its concentration was about 0.0174 � g/mg.

The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity

Potassium tellurite (K 2TeO 3) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO 3 2 - in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 ( aceE), E2 ( aceF), and E3 ( lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.

Reducing, Radical Scavenging, and Chelation Properties of in Vitro Digests of Alcalase-Treated Zein Hydrolysate

The objective of the study was to assess the antioxidant potential of alcalase-treated zein hydrolysate (ZH) during a two-stage (1 h of pepsin --> 0.5-2 h of pancreatin, 37 degrees C) in vitro digestion. Sephadex gel filtration and high-performance size exclusion chromatography were used to separate ZH into fractions. The amino acid composition, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+*)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH*) free radical scavenging activity, reducing power, and Cu (2+) chelation ability were tested to determine the antioxidant efficacy of ZH. Results showed that in vitro digests of ZH contained up to 16.5% free amino acids, with short peptides (<500 Da) making up the rest of the mass. The ABTS(+*) scavenging activity of ZH was decreased by 27% (P<0.05) after pepsin treatment but was fully recovered upon subsequent pancreatin digestion, while the DPPH* scavenging activity of ZH was substantially less than ABTS(+*) scavenging activity and showed a 7-fold reduction following pancreatin treatment. The reducing power of ZH increased 2-fold (P<0.05) following pancreatin digestion when compared with nondigested ZH. The ability of ZH to sequester Cu (2+) was reduced by pepsin digestion but was reestablished following pancreatin treatment. The antioxidant activity demonstrated by in vitro digests of ZH (1-8 mg/mL) was comparable to or exceeded (P<0.05) that of 0.1 mg/mL of ascorbic acid or BHA. The results suggested that dietary zein alcalase hydrolysate may have the benefit to promote the health of the human digestive tract.

Acute in vivo stimulatory effects of human pituitary lipid-mobilizing factor on plasma levels of glucagon and insulin in rabbits.

The lipid-mobilizing factor LMF is prepared from deep-frozen human pituitary glands by alkaline extraction, followed by acetone precipitation at pH 4.8, Sephadex gel filtration and DEAE-cellulose chromatography. In addition to its adipokinetic effect in rabbits, LMF also increased the plasma levels of glucagon and insulin in rabbits in doses of 15 to 100 micrograms. The LMF-induced increases in the plasma levels of glucagon were most pronounced in fasted rabbits, whereas the increases in the plasma levels of insulin were most pronounced in fed rabbits. Glucose infusions decreased the LMF-induced hyperglucagonaemia and increased the LMF-induced hyperinsulinaemia. Somatostatin did not inhibit the LMF-induced hyperglucagonaemia with statistical significance, but inhibited the LMF-induced hyperinsulinaemia. The plasma levels of glucose were slightly decreased by 20 and 40 micrograms LMF in fasted rabbits and were unchanged in fed rabbits. In fasted rabbits, LMF had a toxic effect and 100 micrograms LMF killed one rabbits. Human growth hormone (hGH), prepared from the pituitary glands after removal of LMF, also increased the plasma levels of glucagon and insulin in rabbits. It is possible that the observed effects of LMF and hGH were due to the presence of some biologically active substances from the pituitary gland. These postulated substances could be involved in the pituitary control of the endocrine pancreas, and work is in progress to isolate them from the LMF preparation.

Production, purification and thermal characterisation of invertase from a newly isolated Fusarium sp. under solid‐state fermentation

SummaryProduction of invertase employing a newly isolated Fusarium sp. under solid‐state fermentation was optimised. Different process parameters were optimised. The maximum enzyme activity under optimum conditions was 47.23 ± 2.12 U gds−1 with nitrogen additives. The enzyme was purified by ammonium sulphate precipitation, diethylaminoethyl cellulose ion‐exchange chromatography and Sephadex gel filtration. This protocol gave 20.25‐fold purification and 5.53% recovery. The optimum pH and temperature for activity were 5.0 and 50 °C. The Km and Vmax values for the enzyme were 8.33 mm and 21.48 μmol min−1, respectively. A detailed kinetic study of thermal inactivation has been carried out. Enthalpy of activation (ΔH*) decreased when entropy (ΔS*) of activation increased at higher temperatures. Moreover, free energy of denaturation (ΔG*) increased at higher temperature making the enzyme thermally stable. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed.

Interactions between cells of the immune system and hyaluronate synthesis by human dermal fibroblasts.

Mitogen- or alloantigen-activated human peripheral blood mononuclear cells (MNC) produce a soluble factor which selectively stimulates up to twenty-fold the synthesis of glycosaminoglycan (GAG) by cultured normal human fibroblasts. Confluent fibroblast monolayers were incubated with active MNC supernatants and newly synthesized GAG was measured by the incorporation of [3H]glucosamine into cetylpyridinium chloride-precipitable material. The GAG-stimulatory factor (GAG-SF) was a product of T lymphocytes. Alloreactive T cell clones obtained from the peripheral blood produced the factor after reactivation with the irradiated stimulators, and its production was dependent on HLA-DR-mediated recognition. The CD3+CD4+ clones derived from the skin-infiltrating lymphocytes in patients with early scleroderma also produced the GAG-SF upon in vitro activation with a mitogen. The GAG-SF was purified to apparent homogeneity from supernatants of concanavalin A-activated MNC by Sephadex gel filtration, ion-exchange chromatography and reverse-phase HPLC. The GAG-SF is a 67,000 Da glycoprotein with pI of 5.6. It is not mitogenic to fibroblasts and does not modulate collagen synthesis. Its purification and characterization are important, because of a possible involvement of activated lymphocytes and their products in the immunopathogenesis of human diseases characterized by fibrosis, stromal reactions and local lymphocytic infiltrates.

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Construction and evaluation of anti-gastrin immunogen based on P64K protein

To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect. G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84(th) day after the first immunization, the titer of antibody against cross-linked protein reached 51,200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.

Distribution of plasma-bound hydroxyproline in breast cancer, benign breast disease and healthy females.

In normal adult females, patients with benign breast disease and patients with breast cancer, hydroxyproline (OHPro) was found in the plasma attached to the protein fractions which were separated by Sephadex gel filtration. In addition, a distinct moiety, probably a protein sub-unit, containing substantial amounts of this imino acid, occurred in between gel filtration fractions III and IV. Quantitative differences in the OHPro content of these various fractions were observed in the groups of patients studied. Significant elevations in the peptide-bound OHPro (fraction IV) were found only in patients with breast cancer and the level appeared to correlate with the degree of advancement of the disease. Similar findings relate to the OHPro content of the sub-protein moiety which is found between gel filtration fractions III and IV. A reduced level of high molecular weight hyproprotein (fraction I) was found in the breast cancer group.

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Identification of a novel pulmonary oedema producing toxin from Indian red scorpion ( Mesobuthus tamulus) venom

The experiments were conducted to identify the toxin that produces pulmonary oedema in Mesobuthus tamulus (BT) envenomed animals. Crude BT venom was subjected to Sephadex gel filtration (G-75) and the fractions were screened for optical density (OD), neurotoxicity (prolongation of compound action potential in frog sciatic nerve) and lethality. All these parameters exhibited a peak between 54–94 ml eluates. Fractions of this peak were pooled (SP) and loaded on to carboxymethyl cellulose column. The column was then eluted with increasing buffer concentrations at constant pH and temperature. Eluates were screened for neurotoxicity and OD. Four peaks of neurotoxic activity ( T1– T4) were detected. T2 and T3 were lethal whereas T1 and T4 were non-lethal. T2 exhibited mainly neurotoxicity and failed to augment phenyldiguanide (PDG)-induced reflex response or to produce pulmonary oedema. T3 was having minimal neurotoxic actions but augmented PDG‐reflex and produced pulmonary oedema. The effects of T3 persisted even after dialysis with 8 kDa cut-off filter but not those of T2. The T3 effects resembled toxic manifestations of BT venom and were blocked by aprotinin pre-treatment. T3 demonstrated a band at ∼100 kDa in SDS-PAGE. The results demonstrate the presence of a lethal, high molecular weight, pulmonary oedema producing toxin in BT venom.