Immuno-light and immuno-electron microscopic examination of freeze-dried kidney tissue

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Major outer membrane proteins: common antigens in enterobacteriaceae species

The major outer membrane (OM) proteins of 23 enterobacterial strains (principally clinical isolates) and five non-Enterobacteriaceae species were investigated by the sodium dodecyl sulphate-polyacrylamide gel immunoperoxidase (SGIP) technique to evaluate antigenic cross-reactivity among these proteins. All enterobacterial strains contained one or more peptidoglycan-associated major OM proteins, cross-reactive with the peptidoglycan-bound protein I of Escherichia coli, and one non-peptidoglycan-bound heat-modifiable protein, cross-reactive with protein II of E. coli. Results indicated that antigenic cross-reactivity of the major OM proteins is a general phenomenon in the family Enterobacteriaceae, independent of any molecular weight variation of the corresponding proteins in different bacterial strains. SGIP experiments carried out with OM preparations of other species showed no cross-reactivity of any of their OM proteins with enterobacterial major OM proteins. The significance of the immunological relatedness of OM proteins for the classification of some Enterobacteriaceae is discussed.     

High-throughput identification and screening of novel Methylobacterium species using whole-cell MALDI-TOF/MS analysis

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing.     

Fixation and embedding variables in the immuno-electron microscopic study of rat heymann nephritis

Summary Fixation and embedding variables were compared in immuno-electron microscopic localization of rat IgG in an autologous immune complextype nephritis. Specimens from kidney cortex were fixed for 3, 6 or 9 h in the following fixatives made in 0.1 M phosphate buffer at pH 7.44% paraformaldehyde, 2.5% glutaraldehyde, periodate-lysine-paraformaldehyde or modified Karnovsky's fixative. Localization of IgG was performed on tissue sections cut with a tissue chopper, cryostat or sliding microtome, using agarose, Ames O.C.T. Compound or polyethylene glycol respectively as cutting matrixes. The sections were incubated in peroxidase-labelled antirat IgG antiserum (diluted 120 with phosphate-buffered saline) for 60 h. Peroxidase activity was then revealed and the sections embedded in Epon. Exact localization of IgG throughout the sections and good ultrastructure were achieved when paraformaldehyde and agarose were used. Periodate-lysine-paraformaldehyde proved almost as useful as paraformaldehyde in connection with agarose in respect of peroxidase reaction and ultrastructure. Fixatives containing glutaraldehyde gave a mostly weak and unevenly distributed peroxidase reaction product. In the cryostat sections breaking of the tissue structure could not be avoided. When polyethylene glycol was used as cutting matrix no peroxidase reaction was achieved.     

Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.     

Grouping antigens of four Lactobacillus species and their characteristics.

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2: 1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4: 1. Inhibition tests indicated that the immunodominant component of antigen 9 was a-methylglucoside (glucose), and most probably the determinant is a glycosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

Separation of antigens from Sarcocystis species using chromatofocusing

Crude antigen preparations from bradyzoites of Sarcocystis species exhibit a high degree of cross-reactivity with antisera against heterologous Sarcocystis species, preventing the development of a species-specific immunological test for sarcocystiosis. In this study, we fractionated bradyzoite-derived protein extracts from Sarcocystis tenella, Sarcocystis arieticanis, Sarcocystis gigantea, and Sarcocystis muris by chromatofocusing and obtained distinct protein elution profiles for each species. We then examined the isolated protein fractions for antigenicity with homologous and heterologous reference sera in an enzyme-linked immunosorbent assay. Whereas some antigenic fractions of bradyzoite proteins had equally high reactivity with the homologous and heterologous sera, the reactivity of other fractions was 3-38 times higher with homologous serum than with heterologous sera. Mice immunized with less cross-reactive protein fractions of S. gigantea and S. muris bradyzoites produced a specific immune serum. Thus, it is possible to isolate species-specific antigens from crude mixtures of bradyzoite-derived Sarcocystis antigens for development of species-specific immunological tests for sarcocystiosis.     

Immunofluorescence confocal laser scanning microscopy and immuno-electron microscopic identification of keratins in human materno-foetal interaction zone

We show here that at least 5 keratin proteins are present in villous trophoblast and the same 5 in extravillous trophoblast. A further 14 tested were undetectable in these tissues. In contrast, 10 of the 19 keratins tested were present in amniotic epithelium. The marking of amniotic epithelium on the one hand, as distinct from villous and extravillous trophoblast on the other, can be achieved using 5 keratins (K4, 6, 13, 14 and 17) with a mixture of positive and negative discrimination that is expected, in combination, to be highly sensitive. All the specific keratins identified in trophoblast were apparently up-regulated on the pathway to extravillous trophoblast. Co-ordinated differentiation at the molecular expression level is indicated by this finding. The relevant keratins are K5, 7, 8, 18 and 19. Specific keratins have been identified that are down-regulated in villous trophoblast in pre-eclamptic pregnancy. This difference between healthy and pre-eclamptic chorionic villous trophoblast keratin expression was statistically significant in 4 out of the 5 keratins. This was not the case for the extravillous trophoblast at the immunofluorescence confocal level but significant differences were obtained using immunogold electron microscopy. We suggest that the villous trophoblast in pre-eclamptic placentae is cytoskeletally weaker with respect to the filaments made from these specific proteins and that this is one reason why, in pre-eclampsia, trophoblast is deported in greater quantity than in healthy placentae.     

The ER-TR4 monoclonal antibody recognizes murine thymic epithelial cells (Type 1) and inhibits their capacity to interact with immature thymocytes: immuno-electron microscopic and functional studies

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick vibratome sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.     

Immunoelectron microscopic study of the insoluble antigens of bone marrow cells in rats

An immunoperoxidase study of the localization of insoluble antigens was carried out on the rat bone marrow cells. The effect of different fixatives and inhibitors of endogenous peroxidase on the cell ultrastructure and the preservation of immunoreactivity of the cell antigens. The best results were obtained while fixing with 1% paraformaldehyde and 0.05% glutaraldehyde mixture added with 0.5 saponin and using 10% acetic acid as an inhibitor of endogenous peroxidase. Differences were found in the localization of antigens and the intensity of immunoperoxidase staining in cells of different lines of differentiation and degree of maturity.     

Detection of cross reactive antigens between Pestalotiopsis theae and tea leaves and their cellular location

Among the 12 varieties of tea tested against three isolates of Pestalotiopsis theae, causal agent of grey blight disease, Teen Ali-17/1/54 and TV-23 were found to be highly susceptible while CP-1 and TV-26 were resistant under identical conditions. Leaf antigens were prepared from all the tea varieties, three isolates of P. theae and a non-pathogen of tea (Bipolaris tetramera). Polyclonal antisera were raised against mycelial suspensions of P. theae (isolate Pt-2) and leaf antigens of Teen Ali-17/1/54 and CP-1. These were compared an immunodiffusion test and enzyme-linked immunosorbent assay to detect cross reactive antigens (CRA) shared the host and the parasite. CRA were found among the susceptible varieties and isolates of P. theae (Pt-1, 2 and 3). Such antigens were not detected between isolates of P. theae and resistant varieties, B. tetramera and tea varieties or isolates of P. theae. Indirect staining of antibodies using fluorescein isothiocyanate (FITC) indicated that in cross sections of tea leaves, the CRA was concentrated in the epidermal cells and mesophyll tissues. CRA was present in the young hyphal tips of the mycelia and on the setulae and appendages of the conidia of P. theae.     

Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.     

Molecular forms of lipases and their localization in the fungus Rhizopus microsporus by immuno-electron microscopy

Several lipases differing in their molecular masses (24, 32, 43, 66 and 98 kDa and 28, 40, 45 and 69 kDa) were found in Rhizopus microsporus UzLT-4B and UzLT-5C, respectively. The lipases in each strain were immunologically related. Strain UzLT-5C grown on a medium with lipid substrate secreted lipases of 32, 66 and 98 kDa whereas strain UzLT-4B produced lipases of 45 and 69 kDa on the same medium. Immuno-electron microscopy indicated that the intracellular lipases were in peripheral zones of the hyphae, primarily in the periplasm and adjacent vesicles. Immobilization in Ca-alginate gel revealed unusual structures in the cell wall. These structures accumulated lipases and, apparently, exported the enzymes out of the cell.The authors are with the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142292, Pushchino, Moscow Region, Russia     

High performance liquid chromatography of nucleotides. Major methods and their development

The separation of mono- and oligonucleotides possibilities by means of high performance ion-exchange, reversed-phase, so-called "ion-pair" and adsorption chromatography are studied. The influence of the eluent composition (solvent, salt) and pH on the retention, selectivity and resolution in reversed-phase and ion-exchange chromatography is investigated. The model of the hydrophobic-pair ion-exchange mechanism of ion-pair chromatography is considered. The conditions for analysis and preparative isolation of a desired component are optimized for selectivity, resolution and throughput. The methods for prediction of the optimal gradient elution program reasonable resolution at the desired retention time and for choosing the guard-column packing material are proposed. A design of the gradient for system and the version of slurry packing method for HPLC prolonged life-time columns are improved. The automatized analytical technique for determination of the oligonucleotide monomeric composition with two coupled microcolumns is described, that involves enzymatic digestion of an oligonucleotide followed by ion-exchange separation of the hydrolysate.