Relationship between lectin, alpha-, beta-glucosidase, and beta-galactosidase activities of Azospirillum]

The activities of alpha-glucosidase, beta-glucosidase, and beta-galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol-acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied among the azospirilla studied. The cofunction of the A. brasilense Sp7 lectin and beta-galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum 59b lectin and glucosidases was revealed. The lectin from A. lipoferum 59b may possess saccharolytic activity.     

A Common Role for Target Cell Histocompatibility Antigens in Both Nonspecific and Specific T Lymphocyte-Mediated Cytolysis

We have investigated the role of target cell major histocompatibility complex antigens (MHC-Ag) in nonspecific lectin-dependent lymphocyte-mediated cytolysis (LDCC). In contrast to previous reports, we provide evidence that in LDCC the lectin Concanavalin A (Con A) does not mediate lysis by simply bridging cytotoxic T lymphocytes (CTL) and targets via cell surface sugars or by activating the lytic function of CTLs attached to targets via the lectin. Lysis occurs when target cells are pretreated with lectin, but not when CTL are pretreated. Moreover, when CTL populations are used as both aggressors and targets, and only one is pretreated with lectin, lysis occurs only in the direction of the pretreated CTL target. We have observed that in LDCC, as in specific CTL-mediated killing, target recognition proceeds through interaction of CTL receptors (distinct from sugar moieties) and target cell surface determinants perhaps modified by, but distinct from, the lectin itself. We present evidence that the target determinants recognized in LDCC are MHC-Ag: 1) Cells that display reduced amounts of MHC-Ag are poor targets in LDCC; 2) removal of MHC-Ag by papain renders targets refractory to LDCC, however susceptibility is regained upon regeneration of MHC-Ag; and 3) antisera to target cell MHC-Ag block LDCC. The latter finding is also observed in oxidation-dependent CTL-mediated cytotoxicity. Involvement of MHC proteins in both specific and nonspecific CTL-mediated lysis reconciles an apparent fundamental distinction between these two processes and suggests a possible role for MHC proteins in a postrecognition step(s) leading to lysis.     

分枝杆菌噬菌体整合及裂解的分子机理

结核病仍旧威胁着全球人类健康,中国是结核病高发国家之一,寻求新的药物和疫苗势在必行。随着对噬菌体研究的深入,分枝杆菌噬菌体成为结核病新型药物发现和药敏实验的研究热点之一。噬菌体进入宿主菌体内,以裂解和溶源两种途径进入循环。以分枝杆菌的溶源性噬菌体为例,综述了分枝杆菌噬菌体整合和裂解分子机理。分枝杆菌溶源性噬菌体的整合需噬菌体基因组的附着位点attachment site(attP),宿主菌分枝杆菌基因组的附着位点attachment site(attB),整合酶integrase(Int)和整合宿主因子integration host factor(mIHF)。部分溶源性噬菌体如Ms6进入裂解循环,复制转录组装成新的子代噬菌体,在裂解素(Lysin)和穿孔素(Holin)的协同作用下裂解宿主菌,释放子代噬菌体。目前国内未见对分枝杆菌噬菌体的研究报道。研究分枝杆菌噬菌体整合及裂解机理对结核病治疗新药开发有一定的启示。     

Identification of a lysin associated with a bacteriophage (A25) virulent for group A streptococci.

A phage-associated lysin was found in culture lysates resulting from the propagation of virulent bacteriophage A25 on the group A streptococcal strain designated K56. In contrast to the previously described group C streptococcal phage-associated lysins, A25 phage-associated lysin was more active on chloroform-treated cells, was not phage bound, and was active on some group G and H strains, as well as on group A and C strains. A25 phage-associated lysin had an optimum pH of 6.7 and was inactivated by 10(-3) M p-hydroxymercuribenzoate. Group A cells exposed to penicillin were more susceptible to A25 phage-associated lysin, whereas chloramphenicol-treated cells became resistant to lysis. Release of lipoteichoic acid appeared to precede lysis, and cardiolipin treatment of cells reversed the effects of chloroform and penicillin treatments. These results suggest the possibility that A25 phage-associated lysin may have a mechanism similar to the mechanism of an autolysin or that cell lysis may be due to the activation of an autolysin.     

Cytokines alter target cell susceptibility to lysis. II. Evaluation of tumor infiltrating lymphocytes

We studied the susceptibility of autologous and allogeneic tumors to lysis by human tumor infiltrating lymphocytes (TIL) after pre-incubation of the tumors with human rIFN-gamma and human rTNF-alpha. Preincubation of the tumor lines with IFN-gamma or TNF enhanced susceptibility to lysis significantly; the combination of both cytokines was more effective than either alone. Pretreatment for at least 24 h was required to enhance lytic susceptibility and maximal lysis was observed after pretreatment for 48 to 72 h. Highly specific TIL lysed only their autologous tumor targets and failed to lyse cytokine pretreated allogeneic tumor cells. In TIL populations with varying specificity, cytokine pretreatment of targets enhanced autologous lysis as well as allogeneic lysis. This cytokine-mediated effect could also be observed in a lectin-dependent cytotoxicity assay and did not correlate directly with enhanced expression of MHC class I Ag or the adhesion molecules LFA-3 and ICAM-1. These results suggest that enhancement of lysis may occur at a postbinding stage by making the target cell more sensitive to the cytotoxic factors delivered by the killer cell. The fact that lysis of cytokine treated targets by cells with LAK activity was not enhanced suggests that cells with lymphokine-activated killer activity and tumor-derived T cells kill tumor targets via different mechanisms.     

Phytohemagglutinin (PHA)-resistant Chinese hamster ovary cell mutants acquire sensitivity to the lectin after fusion with liposomes containing PHA receptor glycoproteins

Phytohemagglutinin receptor glycoproteins have been inserted into phospholipid vesicles and these have been fused with phytohemagglutinin-resistant chinese hamster ovary cells. Our results show that the fused cells acquire "neoreceptors" for the lectin phytohemagglutinin. Fluorescence activated cell sorting analyses show that approximately 40% of the cells fused with the receptor-containing vesicles. Studies with 125I-labelled lectin showed that fused cells bound three times more ligand than untreated mutant cells. Furthermore, lectin receptors were functionally inserted in the mutant cell plasma membrane. Fused cells cultured in the presence of lectin (200 micrograms ml-1) lost rapidly (8 hours or less) their ability to incorporate [3H] thymidine. Whereas mutant cells cultured for 16 hours in the presence of 50-400 micrograms ml-1 of lectin remained viable, fused cells showed a 45% decrease in 3H-labelled nucleotide incorporation. The method described here should be of general applicability for the study of lectin-dependent cytotoxicity in chinese hamster ovary cell lines.     

A zone phenomenon and a progressive reaction occurring with a radioactive hemolysin,sodium erucate,I 131

Sodium erucate reacts progressively (i.e., once the reaction is started in a time which is so short that the lysin is in contact with the red cells for 30 seconds, it cannot be stopped even by being diluted 10-fold) with human red cells at pH 7. At the same time, systems containing the lysin and human red cells show a zone phenomenon, lysis occurring most readily in a certain concentration of lysin but more slowly in larger or smaller concentrations. Sodium erucate-I¹³¹ can be used to investigate both the zone phenomenon and the progressive character of the reaction. As regards the former, large concentrations of the lysin react relatively poorly with the red cell surfaces and the resistance of the red cells is relatively high. This may be due to the presence of an admixed inhibitor or to the development of an inhibitory state. The lysin is taken up and fixed by material in the red cell surface, so that the "internal phase" of lysin attached to the cell surfaces is so firmly fixed that a 10-fold dilution has no effect on it. It follows that lysis in these systems is progressive, as it is found to be.     

STUDIES ON THE EGG-MEMBRANE LYSIN OF TEGULA PFEIFFERI: QUANTITATIVE ASSAY OF THE LYTIC ACTIVITY

An egg-membrane lysin was partially purified from the sperm extract of Tegula pfeifferi. A turbidimetric and a chemical method with the use of isolated egg membrane as the substrate have been proposed for the quantitative assay of the lysin. The methods are shown to be valid over a limited range of lysin concentration. The pH optimum for the lytic activity is at about 8.3. The lysin shows considerable thermo-instability and is highly sensitive to PCMB and heavy metals. The reduction in turbidity of egg-membrane suspensions during lysis is accompanied by the release of a substance(s) containing neutral sugar. The possibility that the lytic process is composed of two steps is discussed.     

Characteristics of a Lytic Enzyme Induced by Bacteriophage Infection of Micrococcus lysodeikticus

A lytic enzyme induced in Micrococcus lysodeikticus strain 1 by infection with N1 bacteriophage was purified 45- to 50-fold by ammonium sulfate precipitation, acid precipitation, and selective adsorption of contaminating proteins with calcium phosphate gel. The optimal pH for activity of the enzyme was 6.5 to 7.0. Maximal activity occurred at 45 to 50 C and at an ionic strength of 0.06. The enzyme had a limited specificity and lysed cell walls of M. lysodeikticus with the release of dinitrofluorobenzene reactive groups. Living cells were lysed in the absence of phage; however, the rate of lysis increased when phage was present in excess of 10 particles per bacterial cell. Young cells were most sensitive, and the sensitivity decreased to a minimum with stationary-phase cells. Acting synergistically, lysozyme and the N1-induced lysin caused lysis of cells which were resistant to either enzyme acting independently. The N1 lysin did not exhibit proteolytic activity.     

噬菌体裂解酶研究进展

噬菌体裂解酶是双链DNA噬菌体所特有的细胞壁水解酶。研究表明,所有噬菌体裂解酶在结构上具有相似性,即含有2个结构域：比较保守的N端催化区和差异较大的C端特异性结合区。裂解酶的高亲和性与种属特异的细胞壁糖基有关,而后常常是细菌存活的必要成分。所以,细菌难以产生对裂解酶的抗性。本简要综述噬茵体裂解酶的研究进展。     

Purification and characterization of a novel C-type hemolytic lectin for clot lysis from the fresh water clam Villorita cyprinoides: A possible natural thrombolytic agent against myocardial infarction

Villorita cyprinoides (black clam) is a fresh water clam that belongs as a bivalve to the group of mollusc. The saline extracts from the muscle reveal high titers of agglutination potency on trypsin-treated rabbit erythrocytes. With the help of affinity chromatography a hemolytic protein with lectin activity which could all be inhibited by d-galactose were isolated. The lectins were separated on DEAE-cellulose and the main component was purified after an additional step of gel filtration on sephadex G-75. The main component is a non-glycosylated protein with a molecular weight of 96,560 Da determined by MALDI-ToF, consisting of a single protein chain and characterized by the lack of polymers and intermediate disulfide bonds. The pure main lectin with clot lytic feature shows two bands at molecular weights 36,360 and 26, 520 Da. Optimal inhibition of the pure lectin is achieved by d-galactose containing oligo- and polysaccharides. The lectin activity decreased above 40 °C and was lost at 62 °C, the stability over the pH range between 7.0 and 8.0 and requires divalent cations for their activity. The novel C-type hemolytic lectin for clot lysis from the clam Villorita cyprinoides was identified and evaluated, the purified hemolytic lectin (0.35 mg/ml and 0.175 mg/ml) enhanced clot lysis activity when compared to the different concentration (5 mg/ml and 1 mg/ml) of commercial streptokinase. In the present study identified hemolytic lectin was a rapid and effective clot lytic molecule and could be developed as new drug molecule in future.     

Lectin-dependent localization of cell surface sialic acid-binding lectin Siglec-9

Siglecs are immunoglobulin lectin group proteins that recognize the sialic acid moiety. We previously reported that the expression of Siglec-9 on the macrophage cell line RAW264 markedly enhanced Toll-like receptor (TLR)-induced interleukin (IL)-10 production and inhibited the production of proinflammatory cytokines. In this study, we examined the lectin-dependent anti-inflammatory activities of Siglec-9. IL-10 production was modestly reduced by a mutation that disrupted the lectin activity of Siglec-9, while the reduction in tumor necrosis factor-α was not affected. Membrane fractionation experiments revealed that a part of Siglec-9 resided in the detergent-insoluble microdomain, the so-called lipid raft fraction. The amount of Siglec-9 in the lipid raft fraction rapidly increased following TLR2 stimulation by peptidoglycan and peaked after 3–10 min. This time course was similar to that of TLR2. The double tyrosine mutant in immunoreceptor tyrosine-based inhibitory motifs moved to lipid rafts in a similar manner, while lectin-defective Siglec-9 was not detected in the lipid raft fraction. The production of IL-10 was partially reduced by cholesterol oxidase that disturbed lipid raft organization. Taken together, these results suggest that Siglecs exhibit lectin-dependent changes in cellular localization, which may be partly linked to its control mechanism that increases the production of IL-10.     

Jacalin: an IgA-binding lectin

We previously reported that seeds of Artocarpus integrifolia (jackfruit) contain a lectin, which we call jacalin, that is both a potent T cell mitogen and an apparently T cell-independent activator of human B cells for the secretion of immunoglobulins. During the above experiments we noted a massive precipitation in cell cultures stimulated with greater than or equal to 100 micrograms of lectin. In this paper, we show that the precipitate is formed after the interaction of jacalin and the serum protein added to the culture medium. More importantly, we demonstrate that IgA is probably the major serum constituent precipitated by the lectin and that no IgG or IgM can be detected in the precipitates. In secretions such as colostrum, IgA is the only protein precipitated by jacalin. On the basis of this specificity we describe a simple and reliable affinity chromatography procedure for the purification of both human serum and colostrum IgA. Jacalin is a D-Gal binding lectin and should be a useful tool for studying of serum and secretory IgA.     

Staphylococcal Bacteriophage-Associated Lysin: a Lytic Agent Active Against Staphylococcus aureus

A lytic enzyme active against viable, intact staphylococci is released into culture fluids upon lysis of bacteriophage-infected Staphylococcus aureus PS53 cells. This enzyme, staphylococcal phage-associated lysin (PAL), was partially purified by ammonium sulfate precipitation and gel filtration through Sephadex G-200. PAL is optimally active at pH 6.5 and 30 C, and lytic activity is greatly enhanced by the addition of reducing agents. Lytic activity was observed against all strains of staphylococci tested and against purified staphylococcal cell walls, but no activity was noted against other bacterial species. PAL possesses peptidase activity and results in the production of spheroplasts which can be osmotically stabilized for extended periods by the addition of 7.5% polyethylene glycol 4000.     

Influence of concanavalin a on autolysis of gametes from Chlamydomonas reinhardii.

The phytohemagglutinin concanavalin A inhibited zygote formation of Chlamydomonas reinhardii. 15--50 mug lectin/ml not only interfered with the mating reaction, but also with cell wall lysis of gametes and zoospores in a crude autolysin preparation gained from copulating gametes. Further, the structure of cell walls shed into the medium after autolysis in the course of the mating reaction and after lysis "from without" in the crude autolysin preparation was stabilized by Con A. Therefore, it must be assumed that the lectin inhibited zygote formation of C. reinhardii by interfering with autolysis of the cell walls of the gametes. Though Con A inhibited the lytic processes of C. reinhardii, an activation of the autolytic system in theta gametes by the lectin was found to compete with its inhibitory reaction. Con A induced autolysis of theta gametes was dependent on adherence of the cells by their flagella to the surface of the culture vessel or the liquid medium and did not occur in cultures stirred by rotation. The interferences of Con A with the autolytic serum of C. rienhardii were inhibited by methyl-alpha-D-mannopyrano-side and to a lesser degree by glucose, indicating that the carbohydrate binding sites of the lectin were involved in its reactions with the cells.     

The lysis of gram-negative Alcaligenes eutrophus and Alcaligenes latus by palmitoyl carnitine

Summary The use of synthetic palmitoyl carnitine, naturally occurring in cellular membranes, was investigated for the lysis of Alcaligenes eutrophus and Alcaligenes latus. The optimal concentration of the lysin was 1.0 mM and the lysis was almost completed in 60 minutes. Alcaligenes latus was more susceptible to the lytic activity of palmitoyl carnitine than Alcaligenes eutrophus. Palmitoyl carnitine was found to be a more effective lysin than lysozyme.     

Cell Wall Lysin as a Component of the Bacteriophage ?6 Virion

Cell wall lytic activity was found in particles of the lipid-containing bacteriophage ø6. The activity can be extracted from the virion with Triton X-100 in the presence of salt. This treatment removes the membrane-like envelope of the virion which includes five proteins. The lysin requires detergent for in vitro activity. Virus particles formed in nonsuppressor cells by several classes of ø6 nonsense mutants contained the lysin activity; however, particles formed by a mutant (unable to make proteins P5 and P11) had very low activity; high activity was produced when particles were formed in a suppressor host. A study of the time course of the appearance of the lysin during infection showed that it appeared and increased in cells infected with wild-type virus and in suppressor cells infected with a mutant of class 511, but it did not increase in nonsuppressor cells infected with the class 511 mutant. It is concluded that protein P5 is a component of the lysin and that the role of its activity is in both early and late stages of infection. In particular, the lysin may be necessary for the passage of the infecting core of the virion through the cell wall of the bacterium, as well as in the final lysis necessary for the liberation of progeny phage. A mutant of the virus that produces a larger-than-normal protein P10 does not induce normal lysin activity in host Pseudomonas phaseolicola HB10Y, although it does in strain ERA Pseudomonas pseudoalcaligenes. This indicates that protein P5 is probably not sufficient for lysin activity, but the nature of the interaction between P5 and P10 is unknown.     

Studies on the egg-membrane lysin of tegula pfeifferi the reaction mechanism of the egg-membrane lysin

The mechanism of lysis of egg membrane was studied using pure egg-membrane lysin and the isolated egg membrane of a sea snail, Tegula pfeifferi. Kinetic analysis of the reaction and chromatographic fractionation of the reaction mixture demonstrated that the lysis can be divided into three phases in terms of lysin concentration and product release. At low concentrations of lysin (Phase I), all the lysin added was precipitated with a part of the egg membrane and nothing appeared in the supernatant. At medial concentrations of lysin (Phase II), all the lysin added precipitated and a substance(s) from egg membrane was released into the supernatant. At high concentrations of lysin (Phase III), excess lysin remained in the supernatant along with the soluble product(s) released from the egg membrane at the medial concentrations.Either when or after the lysin reacted with egg membrane, it adsorbed to an insoluble part of egg membrane and lost its activity. The maximal amount of lysin adsorbed is proportional to the amount of egg membrane. The strong bond between lysin and an insoluble part of egg membrane cannot be dissociated by 8 M urea, 0.1 M HCl, 0.1 M KOH or 10^?3 M dithiothreitol.The plot of lysin concentration versus product release showed a sigmoidal curve. In the presence of excess lysin, the amount of the product released is proportional to the amount of egg membrane. The product(s) from egg membrane is a mucopolysaccharide-protein complex(es) with a molecular weight of 5 · 10⁶ or more. Stoichiometric analysis showed that about 2000 (or more) molecules of lysin are required to liberate one molecule of the soluble product.These results strongly indicate that the lytic action of egg-membrane lysin is a stoichiometric rather than an enzymatic reaction.     

Identification of a chicken CLEC-2 homologue, an activating C-type lectin expressed by thrombocytes

Receptors on natural killer (NK) cells are classified as C-type lectins or as Ig-like molecules, and many of them are encoded by two genomic clusters designated natural killer gene complex (NKC) and leukocyte receptor complex, respectively. Here, we describe the analysis of an NKC-encoded chicken C-type lectin, previously annotated as homologue to CD94 and NKG2 and thus designated chicken CD94/NKG2. To further elucidate its potential function on NK cells, we produced a specific mab by immunizing with stably transfected HEK293 cells expressing this lectin. Staining of various chicken tissues revealed minimal reactivity with bursal, or thymus cells. In peripheral blood mononuclear cell and spleen, however, the mab reacted with virtually all thrombocytes, whereas most NK cells in organs such as embryonic spleen, lung and intestine were found to be negative. These findings indicate that the gene may not resemble CD94/NKG2, but rather a CLEC-2 homologue, a claim further supported by sequence features such as an additional extracellular cysteine residue and the presence of a cytoplasmic motif known as a hem immunoreceptor tyrosine-based activation motif, found in C-type lectins such as Dectin-1, CLEC-2, but not CD94/NKG2. The biochemical analyses demonstrated that CLEC-2 is present on the cell surface as heavily glycosylated homodimer, which upon mab crosslinking induced thrombocyte activation, as measured by CD107 expression. These analyses reveal that the chicken NKC may not encode NK cell receptor genes, in particular not CD94 or NKG2 genes, and identifies a chicken CLEC-2 homologue.     

THE EFFECT OF HEMOLYTIC SUBSTANCES ON WHITE CELL RESPIRATION

Substances such as saponin, the bile salts, etc., which produce lysis of red cells also produce cytolysis of white cells from rabbit peritoneal exudates, the arbitrary criterion of their cytolytic effect being their ability to depress the O₂ consumption of the leucocytes. The amount of cytolysis increases regularly as the amount of the added lysin is increased, and sufficiently large quantities of saponin, sodium taurocholate, sodium glycocholate, or sodium oleate are capable of virtually abolishing the O₂ consumption altogether. At the same time, it can be shown that a lysin such as saponin is used up in combining with the white cells in much the same way as it is used up in combining with red cells, and the reduction in oxygen consumption appears to be roughly proportional to the amount so combined. The action of these lytic substances on white cells, in fact, is very similar to their action on red cells, due allowance being made for the fact that the cytolysis of the white cell is probably not an all-or-none process like hemolysis. White cell respiration is also depressed in hypotonic solutions, the respiration being virtually linear with the tonicity.