Bacterial endotoxins: biological properties and mechanisms of action

Endotoxins (lipopolysaccharides, LPS) are agents of pathogenicity of Gram-negative bacteria, implicated in the development of Gram-negative shock. Endotoxin reacts with lipopolysaccharide-sensitive cells producing endogenous mediators such as tumour necrosis factor alpha (TNFalpha). Macrophages are cells mediating the toxic activities of LPS and TNFalpha is the primary mediator of the lethal action of endotoxin. This review article discusses the various mechanisms by which endotoxin hypersensitivity in bacteria-sensitized animals develops. The paper concludes with a discussion on the possible protective effect of carnitine congeners against the lethal action of LPS.     

Immunomodulation by Histoplasma capsulatum products; polyclonal activation and mitogenic effects

The presence of reactive spleen cells to sheep red blood cells (SRBC) in nonimmunized BALB/c mice injected with histoplasmin, the culture filtrate of Histoplasma capsulatum, was monitored for 21 days following inoculation. Polyclonal activation, as evidenced by a sharp increase in the number of anti-SRBC rosetteforming cells (RFC), as well as an enhanced response to heterologous non-cross-reactive erythrocytes from other species, was found in the spleens of these rodents on Days 11 to 13. Elimination of B-cell-derived RFC by the addition of complement indicated that the erythrocyte-binding cells consisted of both T- and B-lymphocytes. An immunosuppressive effect was detected if histoplasmin was injected 2 days before the antigen (SRBC), but could be reversed by injecting the filtrate 30 min prior to the antigen, as is found with polyclonal activators displaying immunosuppressive activity. Histoplasmin also had a mitogenic effect on lymphocyte obtained from the spleen, bone marrow, and thymus similar in magnitude to that produced by lipopolysaccharide (LPS) and concanaval in A. The biological significance of these findings is discussed.     

Structure and mitogenic activities of corn cob heteroxylans

Various structurally different water-soluble (ws) and water-insoluble (wis) heteroxylans have been tested for mitogenic and co-mitogenic activity and shown to differ in their stimulating potency. The ws arabinoglucuronoxylan (ws-AGX) from corn cobs exhibited the highest potency which was comparable to that of the immunomodulator zymosan, whereas the wis-AGX from corn cobs was inactive. Water-soluble derivatives of wis-AGX as well as hydrolytically modified ws-AGX fractions were prepared and tested for their mitogenic and co-mitogenic activity. The results indicate that the disaccharide side chains present only in ws-AGX might be important for expression of the immunostimulatory activity of this xylan.     

Relative mitogenic activities of various estrogens and antiestrogens

The abilities of a variety of estrogens and antiestrogens to stimulate DNA synthesis in the prepuberal rat uterus were compared. One microgram of each compound was administered in vivo via a single intraperitoneal injection. DNA synthesis was assayed in vitro in isolated nuclei 24 h later. The relative mitogenicities of the steroidal estrogens were: 16 alpha-E2 less than 17 alpha-E2 = E3 = 16-EpiE3 less than 16 beta-E2 = 17 beta-E2. The potencies of several nonsteroidal estrogens were also tested. Indenestrol A was as potent at 17 beta-E2, whereas indanestrol and dimethylstilbestrol had weaker activities. The antiestrogens, nafoxidine and 4-hydroxytamoxifen, were both potent stimulators of DNA synthesis. The abilities of an estrogen to stimulate increases in uterine wet weight, DNA polymerase alpha activities, and DNA synthesis in uterine nuclei 24 h after injection were closely correlated. Because the magnitude of the stimulation of DNA synthesis was greatest, its measurement is the most sensitive of these assays of uterotrophic activity.     

Therapeutic activities of PHA-L4, the mitogenic isolectin of phytohemagglutinin

Evidence is presented that suggests that PHA-L4 may exert the therapeutic effects of a theoretical ideal biological response modifier through its ability to do the following: to assist remission induction in certain malignancies, to exhibit direct antitumor cytotoxic effects, to enhance antineoplastic effect of radiation and chemotherapy, to decrease the liability to malignant transformation, to promote differentiation and restore normal growth responses in neoplastic cells, to manifest minimal liability to suppressor activity that would inhibit tumor rejection or antitumor cytotoxicity, to repress graft rejection and graft-versus-host responses and amplify the immunosuppressive effects of other agents in allograft transplantations, to display a direct protective effect against damage from radiation and chemotherapy, to stimulate normal myelopoiesis, to reinforce responses against various infections, to amplify tumor immunogenicity, and to attract mononuclear cells to sites of injection or local application.     

Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities

The discovery of endogenous bioactive peptides has typically required a lengthy identification process. Computer-assisted analysis of cDNA and genomic DNA sequence information can markedly shorten the process. A bioinformatic analysis of full-length, enriched human cDNA libraries searching for previously unidentified bioactive peptides resulted in the identification and characterization of two related peptides of 28 and 20 amino acids, which we designated salusin-alpha and salusin-beta. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Intravenous administration of salusin-alpha or salusin-beta to rats causes rapid, profound hypotension and bradycardia. Salusins increase intracellular Ca2+, upregulate a variety of genes and induce cell mitogenesis. Salusin-beta stimulates the release of arginine-vasopressin from rat pituitary. Expression of TOR2A mRNA and its splicing into preprosalusin are ubiquitous, and immunoreactive salusin-alpha and salusin-beta are detected in many human tissues, plasma and urine, suggesting that salusins are endocrine and/or paracrine factors.     

大肠杆菌FtsZ蛋白原核表达及多克隆抗体的制备与鉴定

为制备特异性抗大肠杆菌丝状热敏蛋白Z(Escherichia coli filamentous thermosensitive protein Z,Ec-FtsZ)多克隆抗体,将Ec-FtsZ基因进行化学合成后连接pET-22b(+)表达载体,构建重组质粒Ec-FtsZ-pET-22b(+)。将重组质粒转化到大肠杆菌E.coli BL21(DE3)中进行Ec-FtsZ原核表达与表达条件优化,以HisTrap层析柱进行Ec-FtsZ的分离纯化,再以孔雀绿法进行Ec-FtsZ GTPase(Guanosine triphosphatase)活性测定。使用纯化的Ec-FtsZ为抗原免疫大鼠制备多克隆抗体,经酶联免疫吸附测定实验(Enzyme-linked immunosorbent assay,ELISA)、Western blotting实验和免疫荧光实验鉴定,抗Ec-FtsZ多克隆抗体效价可达1∶256 000且具有良好的抗原特异性。抗Ec-FtsZ多克隆抗体的成功制备为Ec-FtsZ生物学功能研究和生化检测奠定了实验基础。     

Comparison of intracellular and extracellular mitogenic activity

Endothelial cell-derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat-stable and trypsin-sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen-secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56 degrees C) and were not inactivated by trypsin. Similar to platelet-derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.     

Modulation of macrophage-mediated toxicity to cancer cells in vitro by liposomes and endotoxins

The cytotoxic effect of rat peritoneal macrophages on syngeneic colon cancer cells is modified by the addition of liposomes. The direction and intensity of this modification depends on the concentration of endotoxin in the culture medium.     

Synchrony in Human,Mouse and Bacterial Cell Cultures: A Comparison

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.     

A homotetrameric agglutinin with antiproliferative and mitogenic activities from haricot beans

A homotetrameric agglutinin with a molecular mass of 130 kDa was isolated from seeds of the haricot bean. The agglutinin was isolated using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 200. Haricot bean agglutinin was adsorbed on DEAE-cellulose and Affi-gel blue gel. The hemagglutinating activity of the agglutinin was stable up to 40 degrees C. It underwent a 40% decline when the temperature was raised to 50 degrees C and a complete loss when the temperature was further increased to 80 degrees C. The hemagglutinating activity exhibited a time-dependent loss in activity when the agglutinin was incubated at 100 degrees C for different durations. No activity was discernible when the agglutinin was left at 100 degrees C for 1 min. The activity also underwent a decline in the presence of 500 mM FeCl(3) and CaCl(2). Haricot bean agglutinin manifested a weaker mitogenic activity than concanavalin A toward mouse splenocytes. It exhibited antiproliferative activity toward the tumor cell lines M1 [leukemia], HepG2 [hepatoma] and L1210 [leukemia] cells.     

Bacterial activities in the sediment of Lake Velencei,Hungary

Lake Velencei (Hungary) is one of the westernmost shallow soda lakes, extending from Eastern Europe to the Carpatian basin. The spatial and temporal distribution of the sediment microbiota, the metabolic potential of bacterial communities and the species composition of the genera Bacillus and Clostridium, as well as sulphate-reducing bacteria (SRB) were investigated regarding the close interactions between the lake sediment and the overlaying water column, the special water chemical parameters, and the extensive reed coverage of the lake. Aerobic microbial activities were tested with community-level physiological profiling (CLPP) using BIOLOG microplates. The quantification of the anaerobic fermentative and sulphate-reducing bacteria was done by the MPN (Most Probable Number) method. The cultivation of bacteria adapted to the special physico-chemical characteristics of the lake was carried out employing selective media. Multivariate analysis of CLPP data indicated that the microbial communities of the sediment separated from that of the water and showed seasonal variations of the utilised carbon sources. The results of the MPN demonstrated that the counts of the fermentative and sulphate-reducing bacteria in the reed rhizosphere were about one order higher than in the sediment. Among the isolated bacterial strains, a large number were characterised as facultative or obligate alkaliphilic and also moderately halophilic. The partial sequencing of 16S rDNA of the selected representatives resulted in species of aerobic bacteria, such as Bacillus pseudofirmus, B. halmapalus, B. cohnii, B. (Marinibacillus) marinus, and anaerobes, such as Clostridium putrificum – sporogenes, C. scatologenes, C. bifermentans, Desulfotomaculum guttoideum, Desulfovibrio alcoholivorans, and Desulfovibrio burkinensis.

     

Bacterial siderophores: synthesis and biological activities of novel pyochelin analogues

The synthesis and biological activities of four pyochelin analogues substituted in different parts of the molecule are reported: 5-NHBoc-pyochelin, 3"N-Boc-pyochelin, 3"-nor-NH-pyochelin and neopyochelin II, the enantiomer of natural pyochelin. All these compounds complex iron(III) and transport it at different rates into the cells of Pseudomonas aeruginosa.