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.     

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.     

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.     

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.     

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.

     

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.     

Studies on Manganese uptake in Tilapia mossambica and Salmo gairdnerii

Tilapia mossambica fingerlings, 5 weeks of age, were reared in simulated fresh water medium which was either free of manganese or supplied with manganese at a concentration of 2.5 µg/l. They were fed synthetic diets which were either deficient (2.8 mg Mn/kg dry diet) or supplied with manganese (35.5 mg Mn/kg dry diet). The feeding tests were carried for 10 weeks. Best growth was obtained when the element was supplied in both food and water. When manganese was absent or was supplied only in water or food, the following symptoms were noted: (1) Poor growth, (2) reduced food consumption, (3) loss of equilibrium, and (4) increased mortality. From the results, the daily requirement for manganese by Tilapia for growth and development was calculated to be 1.7 mg/kg live fish.This paper is based on a part of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate College of University of Washington, Seattle, Washington.     

莫桑鼻给非鲫卵黄球结晶体的电镜观察

运用透射电镜观察了莫桑鼻给非鲫卵母细胞内卵黄球的结构;卵黄球由被膜、结晶体及两者之间的非结晶区三部分组成。卵黄球结晶体呈斜方点阵,组成结晶体的卵黄高磷蛋白分子排列呈拟六角形列阵。本文还探讨了卵黄球结晶体形成的可能机制及生物学意义。     

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.     

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.     

Ultrastructural study of arterio-venous anastomoses in gill filaments of Tilapia mossambica

Cell and Tissue Research - Arterio-venous anastomoses (AVA) in gill filaments of Tilapia mossambica exhibit a distinct polarity. Two different types of highly specialized endothelial cells, both of...     

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.