Antigenic differences between endozoites and cystozoites of Toxoplasma gondii

Antigenic differences between the cystozoite and endozoite of Toxoplasma gondii were found using fluorescent antibody staining. Antisera against the cystozoite reacted against only the cystozoite, whereas antisera against the endozoite reacted against both endozoite and cystozoite. Absorption of sera with endozoites removed only positive reactions with endozoites. These findings are the first to suggest antigenic differences between these two forms of Toxoplasma.     

Cytochemical study of different stages in the life cycle of Toxoplasma gondii. I. Amylopectin and lipids in endozoites]

Attempts were made to localize cytochemically acid micopolysaccharides, in addition to sulphate groups of mucosubstances in Toxoplasma endozoites of SS-119 and RH strains with negative results. Amylopectin was found to be the only polysaccharide so far detected in the endozoite stage, using the PAS technique. With RH strain toxoplasmas, the dynamics of amylopectin was followed within 6 days after mouse inoculation. On day 2, toxoplasmas and the host cells of the mouse peritoneal exudate being practically PAS-negative, the amount of amylopectin increased progressively towards day 6. The immune response of the host may be presumably involved in the pattern of this dynamics. Phospholipids were detected in the cytoplasm of intra- and extracellular endozoites of strain SS-119. Little, if any, amount of neutral fat was observed. Of special interest is the increased phospholipid content in the infected host cells.     

The immunocytochemical demonstration of Toxoplasma antigen in the brains of congenitally infected mice

The peroxidase anti-peroxidase immunocytochemical staining method was used to identify Toxoplasma antigen in paraffin embedded sections of the brains of 22 mice congenitally infected with the parasite. Intact Toxoplasma tissue cysts were readily demonstrated in the brain in all cases. In 4 of the 22 infected mice there was evidence of rupture of the cyst wall and/or presence of extra-cystic Toxoplasma antigen. Further support for the extra-cystic location of Toxoplasma antigen was obtained by electron microscopy of reprocessed tissue which revealed endozoites in the area immediately surrounding a ruptured cyst. The possible implications of these findings in relation to the pathogenesis of congenital toxoplasmic meningo-encephalitis are discussed.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.     

Nucleus-targeting domain of the matrix protein (M1) of influenza virus.

The matrix protein M1 of influenza virus A/WSN/33 was shown by immunofluorescent staining to be transported into the nuclei of transfected cells without requiring other viral proteins. We postulated the existence of a potential signal sequence at amino acids 101 to 105 (RKLKR) that is required for nuclear localization of the M1 protein. When CV1 cells were transfected with recombinant vectors expressing the entire M1 protein (amino acids 1 to 252) or just the first 112 N-terminal amino acids, both the complete M1 protein and the truncated M1 protein were transported to the nucleus. In contrast, expression in CV1 cells of vectors coding for M1 proteins with deletions from amino acids 77 to 202 or amino acids 1 to 134 resulted only in cytoplasmic immunofluorescent staining of these truncated M1 proteins without protein being transported to the nucleus. Moreover, no nuclear membrane translocation occurred when CV1 cells were transfected with recombinant vectors expressing M1 proteins with deletions of amino acids 101 to 105 or with substitution at amino acids 101 to 105 of SNLNS for RKLKR. Furthermore, a synthetic oligopeptide corresponding to M1 protein amino acids 90 to 108 was also transported into isolated nuclei derived from CV1 cells, whereas oligopeptides corresponding to amino acid sequences 25 to 40, 67 to 81, and 135 to 164 were not transported into the isolated cell nuclei. These data suggest that the amino acid sequence 101RKLKR105 is the nuclear localization signal of the M1 protein.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Cytochemistry and Ultrastructure of Hyphae and Haustoria of Peronospora parasitica (Pers. ex Fr.) Fr.

The uniform distribution of nuclei, mitochondria, lipid material,protein, and RNA in the hyphae and haustoria of Peronosporaparasitica was demonstrated by staining techniques. Glycogenwas not detected, the only insoluble carbohydrate material detectedby the periodic acid-Schiff reaction being in the fungal wall.The host cell walls reacted more intensely to this stain thanthe hyphal walls. The reaction of haustorial walls varied betweenthe slight staining reaction characteristic of the fungal walls,and the strong reaction of the host cell wall. Callose sheathswere occasionally seen. Fine structure was found to be similar to that of other Oomycetes.Welldeveloped sheaths of a vesicular nature, possibly synonymouswith callose sheaths, were occasionally seen partly surroundinghaustoria.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Summary Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1–7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G₂ phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G₂ phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.This work was supported by the Polish Academy of Sciences as a part of project no 09.7.1.4.5     

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M_r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M_r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M_r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.     

Nuclear changes accompanying cell differentiation in stems of Pisum sativum L.

Summary Nuclear DNA, nuclear protein and nuclear size have been measured in cells of the cortex, pith and vascular tissue from three successive internodes in the stem of Pisum sativum. New techniques of computer-linked cytophotometry were used to measure these parameters simultaneously in both section and squash preparations. In cortical cells no endoreduplicated nuclei were seen in the internodes measured. In cortical cells from the oldest internode measured, a population of large nuclei with the 2C DNA amount was observed which was not present in the younger internodes. In the oldest pith nuclei measured a few 8C nuclei were present, but maturing pith was most characterized by increasing nuclear size and the population of nuclei accumulating with the 4C DNA amount. Polyploid nuclei were present in all of the vascular tissue measured, including the youngest internode. Maturing vascular tissue was also characterized by increasing nuclear size. Nuclear protein measurements demonstrated a close link between nuclear protein and nuclear size and suggest that increased nuclear size, with constant DNA content, may be due to increased nuclear protein. This raises the question of the nature and function of this nuclear protein, perhaps more characteristic of differentiating cells than dividing cells.To whom offprint requests should be sent     

Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer.

A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma. The full-length cDNA was subsequently established by 5' and 3' RACE. Nucleotide sequence analysis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus. The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed. A segment of the cDNA was expressed in E. coli to produce a fragment of the PTX1 protein for the generation of specific antibodies. The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower. Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate. The nuclear staining was greatly reduced in prostate carcinoma. The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence.     

Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears.

Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic integrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2--10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.     

Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody

A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.     

Morphological and biochemical evidence for partial nuclear localization of annexin 1 in endothelial cells.

Using immunofluorescence, an affinity-purified anti-annexin-1 polyclonal antibody showed both cytoplasmic and nuclear staining, whereas antibodies against annexins 2, 5 and 6 labelled almost exclusively the cytoplasm of cultured endothelial cells. This was further confirmed by immunogold labelling and electron microscopy using a monoclonal antibody, annexin 1 being detected close to the plasma membrane, in the cytoplasm, as well as inside the nucleus. Finally, using immunoblotting, purified nuclei were shown to contain annexin 1, which was not removed by EDTA treatment. These data open some new perspectives in the understanding of annexin function, including possible involvement in nucleoskeleton dynamics and regulation of proliferation through cell signalling.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

Fibroblast immuno-diagnosis of cytochrome oxidase (COX) deficiency in mitochondrial disease

We studied cytochrome c oxidase (COX) expression patterns in nuclear and mtDNA gene defects. Using quantitative immunocytochemical assay for COX, heteroplasmic staining was seen in MELAS patients with mtDNA mutations but similar staining variability was seen in control cell lines and nuclear gene defects. All fibroblast lines showed a wide variability in cell-to-cell COX I staining intensity. All 8 patient fibroblast lines had reduced COX staining on immunocytochemistry. In 6 lines reduced protein amount was seen on Western blotting and 7 had low COX activity. This study demonstrates that nuclear gene defects can produce a heteroplasmic appearance on immunocytochemistry.