Identification of cultured cell employing specific substance--with special references of "reserve cells" in uterine cervix

Identification of endocervical "reserve cell", which have been regarded as the origin of squamous cell carcinoma of the uterine cervix, was attempted employing immunohistochemically specific substances. The antigenicity of keratin, squamous cell carcinoma antigen(SCC), epithelial membrane antigen(EMA), tissue polypeptide antigen(TPA), vimentin, secretory component(SC) and placental alkaline phosphatase(PLAP) was investigated in histological preparations as well as cultured cells obtained from primary culture of endocervical tissue. The immunohistochemical findings in histological preparations revealed the following: a strongly positive reaction with TPA, a slightly positive reaction with EMA, a very slightly positive with SCC and PLAP, and a negative reaction with keratin, vimentin and SC. Cultured cells were divided into 4 groups according to their morphological characteristics; among these, small rounded or polygonal cells with a centric single nucleus showed similar immunocytochemical reactions to those of "reserve cells" in histological preparations, indicating that "reserve cell" can be growing in culture. The results obtained suggest that immunohistocytochemical specific substances may be useful to identify cultured cells.     

Modulation of immune system cells by lactobacilli

The influence of lactobacilli on the proliferative potential of immune system cells after the intragastral administration of viable microbial cells and their native filtrates to mice CBA is evaluated. The data have been obtained on the modulating influence of lactobacilli on the formation of T- and B-cell immune response--their role in maintaining homeostatsis and specific features of cell mediated immune reactions after the intragastral administration of virulent Shigella dysenteriae for modeling experimental infection in CBA mice. The mechanisms of the immunomodulating action of lactobacilli on local and systemic reactions of the host as well as realization of the protective properties of lactobacilli against the causative agents of acute enteric infections are discussed.     

The methods for correcting with specific and nonspecific immunomodulators the immunopathological processes arising from the administration of a staphylococcal vaccine]

In mice immunized with staphylococcal vaccine the arresting of graft-versus-host reaction under the influence of small doses of staphylococcal vaccine, hyperimmune antistaphylococcal serum, cyclophosphamide, antilymphocytic serum has been demonstrated. Small doses of staphylococcal vaccine stimulated the production of antibodies to staphylococci and dermal extract in the animals, previously immunized with this vaccine, with the simultaneous suppression of cell-mediated immune reactions to both antigens. Immunosuppressing agents have been found to inhibit humoral and cell-mediated immune response to microbial antigen and dermal extract. No influence of vermox and levamisole on the outcome of the graft-versus-host reaction has been registered; the latter preparation has been found to intensify cell-mediated immune reactions to microbial and tissue antigens.     

Two proteases defining a melanization cascade in the immune system of Drosophila

The melanization reaction is used as an immune mechanism in arthropods to encapsulate and kill microbial pathogens. In Drosophila, the serpin Spn27A regulates melanization apparently by inhibiting the protease that activates phenoloxidase, the key enzyme in melanin synthesis. Here, we have described the genetic characterization of two immune inducible serine proteases, MP1 and MP2, which act in a melanization cascade regulated by Spn27A. MP1 is required to activate melanization in response to both bacterial and fungal infection, whereas MP2 is mainly involved during fungal infection. Pathogenic bacteria and fungi may therefore trigger two different melanization cascades that use MP1 as a common downstream protease to activate phenoloxidase. We have also shown that the melanization reaction activated by MP1 and MP2 plays an important role in augmenting the effectiveness of other immune reactions, thereby promoting resistance of Drosophila to microbial infection.     

Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.     

Immunomodulators of microbial origin enhance cytotoxicity of human mononuclear leukocytes and reduce metastatic progression of Lewis lung carcinoma in mice

Effect of immunomodulators for microbial origin on innate immunity and antitumor system was continued to study. Immunomodificator Immunovac VP-4, purified staphylococcal toxoid and glucosaminyl muramyl dipeptide (GMDP) equally enhanced cytotoxicity of mononuclear leukocytes of peripheral blood of healthy donors. Index of cytotoxicity was 2.78, 2.77 and 2.70 respectively. Reduced metastatic progression of Lewis lung carcinoma in mice was observed after Immunovac VP-4 and GMDP administration. Effectiveness was seen when preparations administered according to schedules including their administration before implantation of the tumor. If preparations were administered number of metastases reduced in 4.4-5.6 times and size of metastases reduced in 7-10 times. Interplay between antitumor activity of studied immunomodulators and cytotoxic activity of NK-cells, which are base effectors of antitumor immune response, are discussed.     

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl-β-d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger β-d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

Biological consequences of fatty acid oxygenase reaction mechanisms

Fatty acid oxygenases catalyze the insertion of molecular oxygen into polyunsaturated fatty acids. The enzymic reactions that have been studied in detail exhibit a continuous requirement for a hydroperoxide activator and appear to proceed by a free radical chain reaction. The self-limiting nature of fatty acid oxygenase-catalyzed reactions appears to be due to enzyme self-inactivation during the reaction rather than to product inhibition. Thus “suicide” substrates are potential regulators of overall enzyme activity, although linoleate is a much weaker inactivator than the highly unsaturated fatty acids. Inhibition by added glutathione peroxidase has demonstrated the need for hydroperoxide activator in the cyclooxygenase reaction catalyzed by prostaglandin H synthase and the lipoxygenase reactions catalyzed by lung, leukocyte, and soybean enzyme preparations. The regulation of cellular hydroperoxide levels may influence the formation of prostaglandins and other autacoids by fatty acid oxygenases.     

Carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1), apically expressed on human colonic M cells,are potential receptors for microbial adhesion

In the human gut mucosa, specialized M cells deliver intact foreign macromolecules and commensal bacteria from the lumen to organized mucosal lymphoid tissues triggering immune responses. M cells are also major sites of adhesion and invasion for enteric pathogens. The molecular features of M cell apical surfaces that promote microbial normal attachment are still largely unknown. We have demonstrated previously that in the human colonic epithelium, carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1) are integral components of the apical glycocalyx which participate in epithelial–microbial interactions. In this study, based on the reactivity of specific monoclonal antibodies and on immunoelectron microscopy, we show that M cells of human colonic solitary lymphoid follicles express CEA and CEACAM1 on the apical surface. Recently these highly glycosylated molecules have been characterized as protein receptors for different bacteria. This leads us to propose a role for CEA and CEACAM1 in the adherence of enteric bacteria to the apical membrane of colonic M cells. We also hypothesize that, unlike colonic enterocytes, M cells lack the defense mechanism that eliminates CEA and CEACAM1 upon microbial binding and which is based on vesiculation of microvillus plasma membrane.     

Properties of urease immobilized on silochrome by means of disulfide bonds

Grafting of SH-groups to the silica surface through the hydrolytically stable Si-C-bond is conducted by gamma-mercaptopropyltrimethoxysilane. After 2,2'-dithiobis-p-nitrobenzoic acid (Ellman's reagent) activation of sulphydryl groups urease of microbial origin was immobilized by these carriers. Certain properties of the preparations obtained were studied. The Km of the enzyme during nonporous silicon aerosil immobilization is shown to remain without considerable changes. The found variations in properties of silochrome-immobilized urease are caused by the diffusion inhibition for the substrate and product of the reaction observed even when the substrate concentration is two orders higher than Km.     

Tuftsin: its chemistry, biology, and clinical potential

Tuftsin is a tetrapeptide, Thr-Lys-Pro-Arg, which resides in the Fc-domain of the heavy chain of immunoglobulin G. The peptide originates from a specific fraction of the parent protein through enzymatic processing. Tuftsin possesses a broad spectrum of activities related primarily to the immune system function and exerts on phagocytic cells, notably on macrophages. These include potentiation of various cell functions such as phagocytosis, motility, immunogenic response, and bactericidal and tumoricidal activities. The features of tuftsin, coupled with its low toxicity, make the peptide an attractive candidate for immunotherapy. Tuftsin's capacity to augment cellular activation is mediated by specific receptors that were identified, characterized, and recently isolated from rabbit peritoneal granulocytes. Tuftsin has been chemically synthesized by a variety of techniques, some of which are adequate for large-scale preparations. A multitude of analogs have also been synthesized and extensively studied for structure-function relationships.     

Kinetic studies of mammalian and microbial cholesterol esterases in homogeneous aqueous solutions

The kinetics of one microbial and two mammalian cholesterol esterases have been examined using a variety of aryl acetates in homogeneous solution. The mammalian enzymes behaved identically but differed somewhat from that of microbial origin. The reactions of all three were not affected by either electronic or hydrophobic characteristics. Taurocholic acid was without effect on the microbial enzyme; at low concentrations it inhibited the mammalian system, but when present in millimolar amounts notable increases in rate were discerned, attributable to the detergent effect on the enzyme.     

Light microscopic detection of PAS-positive substances with thiosemicarbazide in freeze-substituted ovaries of Paspalum longifolium before pollination

Summary Localization of polysaccharides in the freeze-substituted, Eponembedded ovaries of Paspalum longifolium prior to pollination was carried out by periodic acid-Schiff's (PAS), periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-AgPr) and periodic acid-thiosemicarbazide-osmium (PA-TSC-OsO₄) reactions. The specificities of these three reactions were also studied. These three reactions are all effective for light microscopic demonstration of polysaccharides in the filiform apparatus, starch grains in the cells and PAS substance in the micropylar region. Nonspecific staining of the nucleoli of the egg and polar nuclei was observed in the PAS reaction. The PA-TSC-AgPr reaction is very specific for polysaccharides but its overall reaction takes a much longer period of time than the PAS reaction. The PA-TSC-OsO₄ reaction colors the cytoplasm and nuclei of most cells and therefore stains of the cell walls, especially those of the egg cell and synergids, do not stand out clearly. The synergid cytoplasm contains some amorphous polysaccharides and thus it colors even in PAS and PA-TSC-AgPr preparations. In the mature embryo sac, the egg and central cell as well as antipodals are vacuolated but the two synergids have no visible vacuoles under light microscope. Each synergid has a prominent filiform apparatus at the micropylar end, which stains intensely in all three preparations. The walls of the central cell and antipodals adjacent to the nucellar cells have many inward papillae which are also intensely stained in all three preparations. Starch grains are abundant in the ovary wall and usually absent in the nucellus and integuments. They are present in the egg, central cell and antipodals, but not in the two synergids.     

Biosynthesis of 6-keto PGF1alpha by microsomal acetone-pentane powder preparations from hog aorta, ram seminal vesicles, and bovine corpora lutea: properties of same.

Acetone-pentane powders of microsomal rich acetone precipitated fractions, have been prepared from hog aortas, ram seminal vesicles, and bovine corpora lutea. These preparations are all active in converting C14 labelled PGH2 to prostacyclin. The reaction was followed by quantitation of the spontaneous hydrolytic product, 6-keto PGF1alpha. The heat stability, pH optima, reactions with inhibitors, and other properties of these types are discussed. The comparative behavior of the respective enzyme preparations shows that while qualitatively they behave in a similar manner, quantitatively, there are significant differences between them, particularly with respect to heat treatment, and response to inhibitors.     

Studies on cell adhesion and recognition. I. Extent and specificity of cell adhesion triggered by carbohydrate-reactive proteins (glycosidases and lectins) and by fibronectin

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N- acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.     

Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

Identification of a Human Cancer Related Organ-Specific Neoantigen

Human cancers express organ-specific neoantigens (OSNs) which elicit specific cellular immune responses in the cancer patient, as demonstrated by leukocyte adherence inhibition (LAI), an in vitro immune response assay. A purified protein of MW 40,000 (p40) exhibiting OSN (colon specific) activity was cleaved into specific peptide fragments and their partial amino acid sequences determined. This information was used in the polymerase chain reaction (PCR) to obtain a 992 bp cDNA clone (PCR-992) from a human colon adenocarcinoma cell line (LS-180). By comparison of the predicted amino acid sequence of PCR-992 with the known sequence of p40 peptides, PCR-992 was shown to correspond to almost the entire coding region of p40. Nucleotide sequence analysis suggested that the protein was mycoplasmal in origin due to its high A+T content (76%) and the presence of five in frame TGA termination codons; at least two of the latter are actually read as tryptophan, a known feature of mycoplasma translation. We have confirmed this origin by direct isolation of a contaminating mycoplasma species from the LS-180 cell line and demonstration that it could be hybridized with the PCR-992 probe. Northern and PCR analysis of RNA preparations from the contaminated LS-180 cell line showed that p40 was part of the high affinity transport system operon of Mycoplasma hyorhinis (Dudler et al, EMBO J., 7: 3963-3970, 1988). Total protein lysates of Mycoplasma hyorhinis cultivated without animal cells could elicit positive LAI responses when incubated with cancer patient leukocytes but not with normal patient leukocytes. The organ-specific nature of the response was, however, not observed indicating that host cell-mycoplasmal interactions may play a role in determining the organ-specific nature of p40 seen with the LAI. The significance of these findings will be discussed in the context of previous thinking regarding the origin of OSNs.     

Import of bacterial pathogenicity factors into mitochondria

Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane beta-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors.