Cardiotoxin VII4 from Naja mossambica mossambica. The refined crystal structure

The crystal structure of cardiotoxin VII4 from Naja mossambica mossambica was refined to 2.5 A resolution. Fifty ordered solvent sites were localized and included in the refinement. The final R factor is 0.197 (lambda/(2sin theta) less than 5 A; F greater than 3 sigma). The three-dimensional structure is characterized by two beta-sheets. Of particular interest is the two-stranded beta-sheet in the N-terminal region. This shows a large right-handed twist and, though strongly connected to the core of the molecule, and in particular to the C-terminal end, protrudes out of the bulk of the molecule. The segment of four amino acid residues connecting the two strands of this sheet is particularly exposed. It contains an invariant proline residue that has probably an important structural role, and is completely hydrophobic. Two other conserved hydrophobic zones were identified; the largest extends over the second and third loops, on one side only of the molecule. All side-chains of invariant hydrophobic character (except proline residues) belong to one of these three zones. Also discussed are the dimeric assembly and the rather loose packing in the crystal. The three-dimensional structure is compared with that of short and long alpha-neurotoxins. Comparison with two-dimensional nuclear magnetic resonance results on the 68% homologous cardiotoxin CT X IIb shows an excellent overall agreement. A few differences are probably genuine.     

Pharmacological study on phospholipases A2 isolated from Naja mossambica mossambica venom

The pharmacological properties of three phospholipases A2 (CM-I, CM-II and CM-III) purified from Naja mossambica mossambica venom were studied. The order of their catalytic and indirect hemolytic potencies was CM-I = CM-II greater than CM-III. Among them, only CM-III had a direct hemolytic action on the guinea-pig RBC, which was greatly inhibited by heparin. In the chick biventer cervicis nerve- muscle preparation, both CM-II and CM-III caused neuromuscular blockade with a gradual contracture and a decreased sensitivity to ACh and KCl, whereas no complete neuromuscular block was observed with CM-I up to 30 micrograms/ml. In the mouse phrenic nerve-diaphragm preparation, these three PLA2s abolished twitches evoked by indirect stimulation earlier than those by direct stimulation. Contracture was also produced by CM-II and CM-III. However only the latter was inhibited by pretreatment with heparin. These PLA2s caused myonecrosis in the hind-leg muscle of the mouse when injected intramuscularly. From these results, it is concluded that all of these PLA2s are both neurotoxic and myotoxic.     

Sound production in the cichlid Tilapia mossambica Peters

Aquarium-bred adult and juvenile Tilapia mossambica Peters can produce sounds ofvarying frequency, duration and intensity. However, minor environmental disturbancesmay cause the fish to fall silent for long periods. The sounds produced by excited feedingfishes are different from those produced by territorial males and from those emitted by fryswimming in school formation. The frequency of the sounds recorded varied from about1–16 kHz; no data are available on frequencies lower than 1 kHz. The sound producingmechanism consists of a single ventral and two dorsal pharyngeals located in the buccalcavity and provided with numerous small teeth. These teeth have a specially modifieddistal surface area which is already evident in younger fish. Young Tilapia , including3-week old fry, are able to emit sounds as soon as a sufficient number of teeth havedeveloped in the pharyngeal region.     

The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica.

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.     

Monitoring the purification by high-performance liquid chromatography of cardiotoxins from Naja mossambica mossambica using phase-sensitive two-dimensional nuclear magnetic resonance

High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.     

Phospholipases as possible virulence factor in Vibrio parahaemolyticus

Phospholipase C activity was elevated in pathogenic Vibrio parahaemolyticus isolated from patients. Phospholipase A activity was more pronounced in the nonpathogenic V. parahaemolyticus strains isolated from water. Extracts of the strains containing phospholipase C and A activity but no thermostable direct haemolysin (TDH) were capable of producing lesions in guinea pig skin indicating the presence of a toxic factor other than TDH. It is suggested that the toxic factor may be phospholipase C since the purified enzyme from Clostridium perfringens produced a similar reaction in guinea pig skin.     

Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.     

Phospholipases as a factor of pathogenicity in microorganisms

Many microorganisms secrete enzymes which ensure their penetration into the host cells. Phospholipases belong to this type of molecules capable to derange or destroy cell surface membranes. Recent data from in vitro and in vivo studies establish the role of phospholipases as virulence factors. Except direct cytolytic effect the enzymes express effect on some immune responses. The increased incidence of antibiotic resistance needs new therapeutic approaches for treatment of infections. Phospolipases represent a promising target for development of a novel class of therapeutics with divergent mechanism of action.     

Activation of the acidic isoform of phospholipase A2 from Naja mossambica mossambica venom by oleoyl imidazolide requires the cooperative action of two ionizing groups

The acidic phospholipase A2 isoform from the spitting cobra Naja mossambica mossambica is activated irreversibly by treatment with a molar equivalent of oleoyl imidazolide. The kinetics of the chemical modification of the enzyme can also be monitored by measuring the large reduction of tryptophan fluorescence, which is accompanied by a distinct red shift. The addition of a single molar equivalent of oleic acid to the enzyme produces an instantaneous reduction in fluorescence but with a barely detectable red shift, confirming that the response to oleoyl imidazolide results from covalent modification of the protein rather than hydrolysis of the reagent. The pH dependence of both activation and fluorescence reduction by oleoyl imidazolide has an optimum rate near pH 8.0. We propose that long-chain fatty acids and long-chain acyl imidazolides bind at a single activation site and that the reaction of the imidazolides involves two protein residues, one of which is a nonessential histidine residue and the other a primary amino group.     

Pituitary cytology of the teleost fish Tilapia mossambica (Peters)

In the pituitary of Tilapia mossambica, eight cell types were identified on the basis of their staining reactions. the RPD consists of erythrosinophils and PbH positive cells. PbH cells border the NH and give amphipilic reaction to tri- and tetrachrome dyes and Halmi's stain. Erythrosinophils are also stained with acid fuchsin and orange G. The PPD is made up of acidophils and cyanophils. The acidophils are stained with orange G, acid fuchsin and erythrosin. These cells form a palisade zone between the neurohypophysis and the cyanophils. The cyanophils are AB, AF, ATh, PAS and aniline blue positive. They include both thyrotrophs and gonadotrophs and cannot be differentiated. The gonadotrophs may be those which are degranulated and vacuolated in the breeding season. The PI is formed of PAS- and PbH positive cells which lack any definite pattern of arrangement. Apart from the chromophils, scattered chromophobes were seen throughout the adenohypophysis. Occasionally, cells resembling the cyanophils of PPD were noticed in the RPD and PI also.     

Selective loss of binding sites for the iodinated alpha-neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes

Removal of asparagine-linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated alpha-neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of alpha-neurotoxin-I-saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose-rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high-affinity binding sites for alpha-neurotoxin I (kd = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin-receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of alpha-neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d-tubocurarine or alpha-bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine-linked carbohydrate chains.     

Aminopeptidase from the skeletal muscle of fresh water fish Tilapia mossambica

An aminopeptidase from the skeletal muscle of fish, Tilapia mossambica, was partially purified to 96-fold using salt precipitation, ion-exchange chromatography and molecular sieve chromatography. The enzyme showed optimum activity between pH 6.5-7.5 at 43 degrees C and Vmax and Km of 14.36 units/mg and 0.059 mM respectively with alanine beta-naphthylamide as the substrate. The aminopeptidase having a molecular weight of 305 kDa was activated by sulphydryl compounds and Co2+ and inhibited by bestatin, puromycin and metal chelators. Inhibition caused by metal chelators could be reversed by the addition of Co2+. Inclusion of L-amino acids, particularly isoleucine and leucine, in the assay medium caused inhibition of the enzyme activity. Substrate specificity together with inhibition and activation pattern indicated that the enzyme is alanine aminopeptidase.     

1H nuclear-magnetic-resonance studies of the three-dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.     

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.     

Source of a new crop of oocytes inTilapia mossambica

Summary 1. In the teleostTilapia mossambica (Peters), new crops of oocytes arise from nests of cells of the germinal epithelium as well as from the epithelial strands ramifying into the ovocoel.2. There is no evidence to indicate that degenerating follicle cells form a source for a new crop of oocytes.3. After one spawning is over, the follicular layer of the mature unspawned ova alone undergo atresia.4. Neither immature oocytes nor oocytes in the growth stages undergo degeneration.5. The occurrence of immature, maturing, and fully ripe ova in the ovary at a particular time account for asynchronism inTilapia mossambica.

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Origine des poussées ovocytaires chezTilapia mossambica

Extrait L'ovaire deTilapia mossambica a été étudié dans le but de préciser l'origine des poussées ovocytaires. Après la ponte l'ovaire n'est pas vide pour autant, et il y subsiste des ovocytes à tous les stades du développement. Les replis de l'épithélium germinatif et les tractus épithéliaux ramifiés contiennent des flots de cellules qui produisent des ovogonies primaires, lesquelles se transforment en ovocytes et en cellules folliculaires. Il ne semble pas qu'une nouvelle poussée ovocytaire puisse se faire à partir des résidus des follicules des ovocytes atrétiques. Seuls les ovules mûrs non évacués par la ponte subissent une atrésie et se résorbent; les ovocytes immatures ou en voie de croissance ne dégénèrent pas. L'asynchronisme deT. mossambica se traduit par la présence simultanée, dans l'ovaire, d'ovocytes à tous les stades de croissance et de maturation.