Lytic Factor Research Articles

The release of carbohydrates during mating in Chlamydomonas reinhardi

During mating of gametes of the alga Chlamydomonas reinhardi, certain physiological changes occur. These changes include the formation, in the (+) gamete, of a fertilization tubule and the release, in both mating types, of a lytic factor which removes cell walls. We have been able to quantitate the latter process, by measuring accumulation of released carbohydrates during the mating process. Contrary to published reports, we have found that glutaraldehyde-fixed gametes and isolated gametic flagella, though they cause the agglutination reaction, do not induce the lytic factor. This implies that induction of lysis takes place during the one-to-one (+) and (-) pairing that occurs subsequent to the initial massive agglutination.

Morphological study of lesions induced by snake venoms (Naja naja and Agkistrodon piscivorus) in the lung and cremaster vessels of rats.

The morphological effects of two snake venoms, N. naja and A. piscivorus, and of the Direct Lytic Factor and Phospholipase-A, compounds purified from N. naja crude venom, were investigated on lung and cremaster vessels of rats. The microcirculation of the rat reacts to these two venoms differently: N. naja produces congestion, haemolysis and increased vascular permeability, whereas A. piscivorus causes these alterations, plus haemorrhage and thrombosis. Direct Lytic Factor elicits reponses similar to the N. naja venom but Phopholipase-A has no effect on the vessels. Phospholipase-A does not seem to potentiate the effects of Direct Lytic Factor on the cremaster vessels. The colloidal carbon technique showed that intrathroacic administration of N. naja venom results in a generalised permeability increase of pleural and subpleural capillaries and A. piscivorus injections provoke only carbon retention in capillaries at sites of localised haemorrhage. In cremasters treated with N. naja venom the carbon blackened the venules predominantly but in cremasters in which A. piscivorus venom was administered the carbon particles labelled both venules and capillaries. There was evidence that the main vascular action of the venoms is local and not systemic, that the permeability factors involved in these lesions are different and that the cremaster vessels are much more sensitive to these snake venoms than the pulmonary vessels. Electron-microscopic studies showed mesothelial and epithelial lesions in lungs and an early inflammatory reaction in the cremaster vessels with both venoms. Erythrocyte fragmentation was a constant feature in all vessels. Endothelial degeneration and capillary disintegration in lung and cremaster vessels were observed in animals treated with A. piscivorus venom.

EXTRACELLULAR LYSIS OF THE BLUEGREEN ALGA PHORMIDIUM LURIDUM BY BDELLOVIBRIO BACTERIOVORUS1

SUMMARYWhen either cells of the bacterium, Bdellovibrio bacteriovorus (Stolp & Starr), strain 15143, or a heat‐resistant lytic factor derived from these cells is added to viable cultures of Phormidium luridum var. olivacea Boresch all the algal cells underwent gradual lysis. This effect was obtained with a mean initial bdellovibrio:algal cell ratio of 7.5:1. When P. luridum was mixed with the bdellovibrio cultures the algal chlorophyll content showed an 8‐fold decrease. Concomitantly, this interspecies interaction caused, a 75% inhibition of algal photosynthesis after‐4 h. Heat, treatment of the B. bacteriovorus culture supernatant fluid increased its ability to inhibit photosynthesis approximately 14%. Light, microscopy showed pale granules and intracellular spaces to form in the P. luridum within 16 h after adding the bdellovibrio lytic factor. Subsequent morphological changes included the development of large intracellular spaces, intercellular spaces, spheroplast formation and finally Complete lysis of the algal cells.

Etude ultrastructurale sur la pathogénie de l'invasion du muscle strié par des tumeurs transplantables

en The ultrastructural aspect of the mode of invasion of muscle tissue by N.S. 104 rhabdomyosarcoma, Novikoff hepatoma, and Walker tumor transplanted in rat skeletal muscle is described. The initial process of muscle invasion by these tumors is active intercellular and transcellular infiltration (infiltration through the cell) of myofibers by the tumoral pseudopods and cells. The transcellular infiltration of myofibers begins after the localized dissolution of external lamina in direct contact with healthy neoplastic cell surfaces. The local disappearance of external lamina in direct contact with viable cancer cells suggests the active release of lytic factors in the contact areas by the invading tumoral cells. The destruction of myofibers in areas of invasion occurs sometimes by fragmentation and sometimes by tumoral blockade. It appears to be a secondary phenomenon which depends on the infiltration of viable tumoral cells. Our ultrastructural findings on the pathogenesis of tumoral invasion of skeletal muscle support the principal role of the tumor cell mobility in active tumoral infiltration. However, the dissolution of the external lamina of the invaded myofibers facilitates this infiltration and suggests also the participation of a lytic factor which is released probably by the viable cancer cells in the areas of invasion. These two factors seem to be essential to explain the mechanism of in vivo tumoral invasion.

Mechanism of hemolysis induced by nematocyst venom: Roles of phospholipase A and direct lytic factor

Pure venom from the acontial nematocysts of the sea anemone Aiptasia pallida exhibited phospholipase A (phosphatide acyl-hydrolase; EC 3.1.1.4) activity on a mixture of free phospholipids. Diethyl aminoethyl cellulose fractionation of the venom gave four distinct protein peaks with the phospholipase A activity being restricted to fractions III and IV. These two fractions tested separately also were able to lyse red blood cells weakly. Fractions I and II enhanced the hemolytic activity of fractions III and IV, with fractions I and III giving as much as ninefold enhancement over that of III alone. Fraction I appears analogous to the direct lytic factor of some snake and bee venoms. Fraction III, which could not appreciably hydrolyze the phospholipids of the intact red cell membrane, was able to do so in the presence of fraction I. The sequential interactions of these two nematocyst venom proteins with the red blood cell membrane to produce hemolysis is discussed.

A study of the high molecular weight hemolysin from the skin secretion of the ambhibian Bombina variegata

A. Mar and H. Michl. A study of the high molecular weight hemolysin from the skin secretion of the amphibian Bombina variegata. Toxicon 14, 191–195, 1976.—The high molecular weight hemolysin in the toxin of Bombina variegata was characterized by some chemical and physico-chemical properties and compared with other hemolysins of animal or microbial origin. It is markedly different from snake venom direct lytic factors by its molecular weight and acidic character. The time course of hemolysis is sigmoidal and recalls the reaction kinetics of nematocyst hemolysins ( Hessinger and Lenhoff, Archs Biochem. Biophys. 159, 629, 1973a). Free SH-groups are not responsible for the binding to the cell surface. The activity of the hemolysin depends on Ca 2+ ions.

Ceruleotoxin: An acidic neurotoxin from the venom of Bungarus caeruleus which blocks the response to a cholinergic agonist without binding to the cholinergic receptor site

The toxins from the venoms of Elapid snakes have been extensively studied during the past few years and several of them are currently used to label pharmacological receptors and other membrane bound proteins [ 1,2] . On the basis of their structure and of their pharmacological action, Lee [ 1,3] has distinguished in these venoms the cardiotoxins, which, like the y-toxin, the direct lytic factor or cobramine B, produce cardiovascular alterations responsible for local necrotic lesions [4] , and the neurotoxins which block synaptic transmission. The neurotoxins themselves can be subdivided into two main categories : 1) the a-neurotoxin, such as a-bungarotoxin, naja toxin or cobrotoxin are basic polypeptides of 60&62 or 70-74 amino acids, which block synaptic transmission at the neuromuscular junction like curare [5] and bind with a high affinity to the nicotinic receptor site [2,5] and, 2) the fl-neurotoxins, such as P-bungarotoxin and some phospholipases A have a presynaptic action and block the release of acetylcholine [ 1,6,7]. In this letter, we describe the purification and some of the structural and pharmacological properties of a toxin which does not fall into any of these categories. This toxin, that we shall refer to as ceruleotoxin, is present in the venom of the snake Bungarus caemleus. It is an acidic protein which blocks the depolarisation of Electrophoms electroplaque by cholinergic agonists, even though it does not significantly bind to the nicotinic receptor site. 2. Materials and methods

The complete amino acid sequence of a cardiotoxin from the venom of Naja naja (Cambodian Cobra).

The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.

The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra).

The complete covalent structure of a small, basic protein with cardiotoxic activity is described. This has been isolated from the venom of Naja nigricollis by gel filtration on Sephadex G-75 and gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin, molecular weight 6806 from amino acid composition, consists of 60 amino acids, cross-linked by four disulfide bridges, connecting 3-21, 14-38, 42-53, and 54-59. The protein contains one residue of tryptophan, phenylalanine, and glutamic acid, two residues of arginine and tyrosine, four residues of methionine, and nine residues of lysine. Histidine is absent. The chymotryptic peptides of the oxidized and S-carboxymethylated protein were isolated by gel filtration on Sephadex G-25 and zone electrophoresis on a cellulose column. The sequence was determined by Edman degradation, using the (manual) direct phenylthiohydantoin method and with the use of carboxypeptidase A. Disulfide pairing was determined on thermolysin cleaved peptides from the native protein. The sequence is shown to be homologous to other cardiotoxins and a lytic factor from snake venoms and also shows homology, both in sequence and disulfide pairing to neurotoxins. A partial reduction experiment in the absence of denaturing agent using 14-C-labeled iodoacetic acid as S-carboxymethylating agent shows that disulfide bonds 14-38 and 42-53 were reduced fastest followed marginally by 54-59, and then bond 3-21.

Zona pellucida denudation, blastocyst proliferation and attachment in the rat

The mode of zona pellucida denudation and blastocyst proliferation and attachment was investigated in Wistar rats during day 5 of pregnancy and in ovariectomized pregnant animals given 5-0 mg progesterone daily until the 10th day, when 0-2 mug oestradiol was injected together with progesterone. During day 5 of pregnancy, when only 5 empty zonae were detected from 32 animals, zona denudation occurred by lysis, which took 6 h. Zona denudation occurred by shedding in the ovariectomized pregnant animals; 190 empty zonae were detected from 40 animals. Oestradiol administration to the experimental animals caused lysis of the shed zonae 24-30 h after oestradiol treatment. Air-dried preparations of the embryos showed that the blastomeres proliferated exponentially from a mean of 27-52 +/- 4-0 cells per embryo at 12.00 h on day 5 of pregnancy to 53-96 %/- 2-58 cells per embryo at 22.00 h, with a cell doubling time of 10 h in normal animals. In the ovariectomized animals, blastocysts were arrested at about the 100-cell stage on the 10th day. Oestradiol administration to the experimental animals did not induce mitosis; the embryos implanted without further cell division. The number of free embryos recovered declined from a mean of 12-0 per animal at 12.00 h to 4-25 per animal at 22.00 h on day 5 of pregnancy. The number of embryos recovered from the ovariectomized animals varied between one and 11 per animal. The results show that cell division continued in embryos within ovariectomized pregnant animals in the absence of oestrogen, until about the 100-cell stage. There was no further cell division before nidation when oestradiol was administered. The empty zonae were, however, lysed between 24 and 30 h after oestradiol treatment. The zona lytic factor appears to be oestradiol dependent and of maternal origin. A causal relationship exists between zona lysis and embryo attachment, which is independent of the number of cells per blastocyst.

A volatile factor inducing transmissible lysis in Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker.

Filtered water extract of Gabalong soil with a recent history of take-all in wheat caused lytic plaques to form in agar cultures of a virulent strain of Gaeumannomyces graminis var. tritici. The plaques resembled those produced by Bdellovibrio on plate seeded with bacteria. However, there was no evidence of the presence of bacteria, viruses, or mycoplasmas. The lytic factor was transmissible in culture filtrates to fresh subcultures of the fungus. Exposure of young healthy colonies to sublethal doses of ultraviolet light also induced transmissible lysis. The lytic factor was heat-stable, passed through a 25-nm filter, and was not affected by nuclease (enzymes) or severe irradiation with UV light. It also induced bysis in several other strains of G. graminis. Lysis was always preceded by a growth-stimulatory effect on the fungus. The lytic factor was active as a volatile chemical which induced transmissible lysis and continued to be formed, apparently as a self-perpetuating agent, in lysing cultures of the fungus.

The effect of leukocyte hydrolases on bacteria

Acid hydrolases of human blood leukocytes are highly lytic toStaph. albus, Staph. aureus, andStrep. faecalis. On the other hand, group A and viridans streptococci, encapsulated staphylococci, a variety of Gramnegative rods, andMyc. smegmatis are highly resistant to lysis by leukocyte extracts. The lytic effect of the leukocyte extracts can be mimicked by an artificial "cocktail" which contains crude trypsin, lysolecithin, phospholipase C, and lysozyme. This enzyme mixture is lytic to certain Gram-negative bacteria and encapsulated staphylococci which are resistant to lysis by leukocyte enzymes. Both the leukocyte lysates and the artificial cocktail are more lytic to bacteria harvested from the logarithmic phase of growth than to older cells.Staph. albus andStrep. faecalis, which are not lysed to any appreciable extent by extracts of rabbit intestines, lymphocytes, and platelets, undergo extensive lysis upon the addition of lysozyme, indicating that these cells contain preparatory prolytic agents which are activated by lysozyme. On the other hand, the lysis ofStaph. aureus by extracts of all these cells is less dependent upon lysozyme, indicating that other non-lysozyme-dependent lytic factors are involved in the lysis of this microorganism by certain tissue extracts. It is suggested that the resistance to lysis by leukocyte enzymes of bacterial cell-wall constituents may contribute to the pathogenesis of chronic sequellae, and that artificial enzyme cocktails be used for in vivo treatment of certain chronic inflammatory processes induced by bacteria.

Enhancement of the cobra venom direct lytic factor by prostaglandins and related synergistic phenomena on pulmonary microvascular damage: Bonta, I. L., Van Dijk, M., Noordhoek, J. and Vincent, J. E., Department of Pharmacology, School of Medicine, Erasmus University, Rotterdam, The Netherlands

Enhancement of the cobra venom direct lytic factor by prostaglandins and related synergistic phenomena on pulmonary microvascular damage: Bonta, I. L., Van Dijk, M., Noordhoek, J. and Vincent, J. E., Department of Pharmacology, School of Medicine, Erasmus University, Rotterdam, The Netherlands

The amino-acid sequence of a polypeptide from the venom of Dendroaspis viridis.

The primary structure of a major polypeptide component from the venom of Dendroaspis viridis has been determined unambiguously. The molecule (60 amino acids, four disulphide bridges) closely resembles a relatively non‐toxic component of the venom of Dendroaspis angusticeps. The pharmacological effects of neither of these are understood. The component from Dendroaspis viridis does not block neuromuscular transmission in frogs, is without effect on the excitatory or inhibitor actions of acetylcholine on snail neurons and does not appear to be a lytic factor.

Immune Hemolysis and a Basic Peptide from Cobra Venom

Summary Sheep erythrocytes sensitized with antibody and the first seven components of complement (EAC1-7) undergo rapid hemolysis in the presence of the eighth component of complement (C8) and a basic polypeptide (direct lytic factor, DLF) isolated from cobra venom which synergistically reacts with phospholipase A in hemolysis. EAC1-7 cells are not lysed by either C8 or DLF alone. DLF reacts after the reaction of C8 on EAC1-7 and not before it. These results suggest possible phospholipase activity in activated C8 molecules.

Enhancement of bee venom phospholipase A 2 activity by melittin, direct lytic factor from cobra venom and polymyxin B

Enhancement of bee venom phospholipase A 2 activity by melittin, direct lytic factor from cobra venom and polymyxin B

Kinetics of prolytic events during red cell lysis induced by sea anemone nematocyst venom

1.1. Kinetic studies of the prolytic (induction) phase of hemolysis caused by nematocyst venom isolated from the sea anemone Aiptasia pallida show that two sequential events occur which are affected by venom and Ca2+ concentrations respectively.2.2. The first event, called t1, is affected by venom concentration and has two sub-phases: a rapid one and slow one. These suggest a differential rate of binding of the two known lytic factors of venom.3.3. The seond phase, t2, involves the interaction of Ca2+ with a complex formed by the venom proteins bound to the red cell membrane.

Thermal and Ionic Factors in the Ultraviolet Photolysis of Plant Cell Membranes

AbstractThe exposure of red beet root tissue to ultraviolet (254 nm) at 2 × 106 erg × cm−2× min−1 (0.2 J × cm−2× min−1) causes release of betacyanin after a 20 minute induction period. Ultraviolet‐photolysis is temperature‐sensitive having a thermal threshold at about 10°C. Reduction in pigment release was effected by chlorides of Mg, Ca and Sr, but not by Li, Na or K. This effect was marked but not complete, even at 40 mM concentration.It is concluded that photolysis is indirect, and involves a lytic factor, possibly an oxidant, derived from an original photochemical product.

Osmotic and non-osmotic components of the haemolysis induced by the direct lytic factor (DLF) of cobra venom.

The effect of increasing the (colloid-)osmotic pressure in the extracellular medium on haemolysis by the direct lytic factor of cobra venom (DLF) and phospholipase A has been investigated. For comparison, N-ethyl-maleimide (NEM) and p-chloromercuribenzoate (p-CMB) were used. Dextran and sucrose abolished the haemolytic effect of NEM and p-CMB but reduced only slightly (dextran) or not (sucrose) the weak lytic activity of DLF. Haemolysis by phospholipase A in the presence of DLF, NEM or p-CMB was not significantly inhibited. Hypertonic NaCl solution considerably retarded the onset of haemolysis by DLF plus phospholipase A. The mean corpuscular volume of guinea-pig red cells increased slightly but definitely during incubation with DLF. It is concluded that the haemolytic effect of DLF has non-osmotic as well as osmotic components, and that phospholipase A causes non-osmotic haemolysis. The retardation of haemolysis by hypertonic NaCl probably indicates specific inhibition of bee venom phospholipase A2, not protection of the erythrocytes from osmotic stress.

Differences in binding of the direct lytic factor (DLF) of cobra venom (Naja naja) to intact red cells and ghosts

The binding of direct lytic factor (DLF) from cobra venom (Naja naja) to intact guinea-pig red cells and to guinea-pig ghosts was estimated quantitatively by bioassay of DLF in the supernatant.