Identification and characterization of variants of Crithidia luciliae with altered morphological and surface properties

Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.     

Crithidia luciliae: starvation for purines and/or phosphate leads to the enhanced surface expression of a protein responsible for 3'-nucleotidase/nuclease activity

It has been shown previously that starvation of the trypanosomatid protozoan Crithidia luciliae for purines and/or inorganic phosphate results in increased levels of a surface membrane-associated 3'-nucleotidase/nuclease (3'-N'ase) activity which hydrolyzes both 3'-ribonucleotides and nucleic acids, thereby permitting the organisms to transport these essential nutrients across their cell membranes. A polypeptide with the requisite catalytic properties has been identified by an in situ gel activity assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In current studies, differential synthesis of the protein responsible for the 3'-N'ase activity was not demonstrable by comparisons of SDS-PAGE patterns of nutrient-replete or purine-starved parasites metabolically labeled with either [35S]methionine, [3H]leucine, or [3H]tyrosine. However, surface labeling of nutrient-replete and purine-starved cells revealed the enhanced expression of an 125I surface-labeled 43-kDa protein which comigrated with the 3'-N'ase activity in one- and two-dimensional electrophoretic systems. The amount of this surface-labeled peptide correlated with the level of 3'-N'ase activity as measured by test tube assay. Refeeding adenosine to purine-starved cells led to the loss of both the enzyme activity and the surface iodinatable 43-kDa band as a result of renewed cell division. Starvation of these organisms for phosphate also led to the enhanced expression of the 43-kDa radioiodinatable band. The results indicated that the 3'-N'ase protein, itself, is differentially expressed at the cell surface under conditions which lead to increased enzyme activity.     

Leishmania donovani: regulated changes in the level of expression of the surface 3'-nucleotidase/nuclease

Leishmania donovani promastigotes have previously been shown to possess a surface membrane bound 3'-nucleotidase/nuclease (3'-N'ase) capable of hydrolyzing both nucleic acids and 3'-ribonucleotides. The specific activity of the 3'-N'ase was increased following transfer of the parasites to fresh, nutrient-replete media or to media lacking purines and/or inorganic phosphate (Pi). In nutrient-replete media, the enzyme activity was transiently elevated during the lag and early logarithmic phases of the growth curve; enzyme activity fell as the cells continued into late log and stationary phases. Purine- and Pi-starved cells exhibited significantly greater levels of 3'-N'ase activity than nutrient-replete cells. These levels remained elevated as long as the organisms were maintained in the deficient media. Nutrient-replete and purine-starved 125I surface-labeled parasites displayed differences in electrophoretic patterns. Upon purine starvation, incorporation of radiolabel was increased in proteins which migrated with apparent molecular weights of 70, 43, and 40 kDa. Comigration, in both one- and two-dimensional systems, of 3'-N'ase activity with the radiolabeled 43-kDa band demonstrated that this band was the catalytically active protein. Peptide mapping of the 70-, 43-, and 40-kDa proteins failed to demonstrate similarities in peptide sequence consistent with either a degradation or a precursor/product relationship. Treatment of the 43- and 40-kDa peptides with N-Glycanase indicated that they were differentially glycosylated. The cumulative results of these studies indicated that L. donovani can respond to altered culture conditions by the differential expression of surface proteins. In particular, the differential expression of the protein responsible for 3'-N'ase activity is consistent with the role of this enzyme in purine acquisition.     

Tn5-induced and spontaneous switching of Sinorhizobium meliloti to faster-swarming behavior

Tn5 mutants of Sinorhizobium meliloti RMB7201 which swarmed 1.5 to 2. 5 times faster than the parental strain in semisolid agar, moist sand, and viscous liquid were identified. These faster-swarming (FS) mutants outgrew the wild type 30- to 40-fold within 2 days in mixed swarm colonies. The FS mutants survived and grew as well as or better than the wild type under all of the circumstances tested, except in a soil matrix subjected to air drying. Exopolysaccharide (EPS) synthesis was reduced in each of the FS mutants when they were grown on defined succinate-nitrate medium, but the extent of reduction was different for each. It appears that FS behavior likely results from a modest, general derepression of motility involving an increased proportion of motile and flagellated cells and an increased average number of flagella per cell and increased average flagellar length. Spontaneous FS variants of RMB7201 were obtained at a frequency of about 1 per 10,000 to 20,000 cells by either enrichment from the periphery of swarm colonies or screening of colonies for reduced EPS synthesis on succinate-nitrate plates. The spontaneous FS variants and Tn5 FS mutants were symbiotically effective and competitive in alfalfa nodulation. Reversion of FS variants to wild-type behavior was sporadic, indicating that reversion is affected by unidentified environmental factors. Based on phenotypic and molecular differences between individual FS variants and mutants, it appears that there may be multiple genetic configurations that result in FS behavior in RMB7201. The facile isolation of spontaneous FS variants of Escherichia coli and Pseudomonas aeruginosa indicates that switching to FS behavior may be fairly common among bacterial species. The substantial growth advantage of FS mutants and variants wherever nutrient gradients exist suggests that switching to FS forms may be an important behavioral adaptation in natural environments.     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

Stimulation of the Clonal Growth and Differentiation of Feeder Layer Dependent Mouse Embryonal Carcinoma Cells by β-Mercaptoethanol

Embryonal carcinoma cells are the undetermined stem cells of teratocarcinomas. Supplementation of culture medium with β-mercaptoethanol permits the feeder layer independent clonal growth and differentiation of normally feeder layer dependent embryonal carcinoma cell lines. Differentiated cells within the clones appeared less than 6 days after plating and were distinguished from embryonal carcinoma cells by their morphology, lack of histochemically detectable alkaline phosphatase activity, and secretion of plasminogen activator. Over 70% of the colonies secreted plasminogen activator after 6 days.
In comparison, a different embryonal carcinoma cell line which has lost the potential for substantial differentiation, either in vitro or in vivo forms very few clones (< 1%) which secrete plasminogen activator. Embryonal carcinoma cells derived from the rare clones which secrete plasminogen activator have the same frequency of production of plasminogen activator secreting colonies as the parental cell line.     

Induction of motility and alteration of surface membrane polypeptides in lymphocytes by contact with autologous and allogeneic fibroblasts

Contact with cultured fibroblasts induced and maintained motile behavior in autologous and allogeneic human lymphocytes. After 3 h of contact with fibroblasts, 50 +/- 19% of the autologous lymphocytes were motile and after 24 h the corresponding figure was 49 +/- 18%. On a plastic surface the number of motile lymphocytes in the same individuals generally persisted below 15%. SDS-PAGE of iodine-labeled lymphocytes indicated that contact with fibroblasts but not with plastic for a 3-h period caused the appearance of a 300-kda band and the disappearance of several bands of lower molecular weight. During the course of T-lymphocyte activation by concanavalin A or allogeneic cells on a plastic surface, the number of motile forms did not reach a maximum (30 to 50% in separate individuals) until after 2 to 4 days in culture. Thus, in terms of both rate of development and number of motile forms, the fibroblast-dependent motility mechanism was more effective than conventional lymphocyte activation to blast transformation. Conditioned medium from fibroblasts did not induce motile behavior in the lymphocytes and did not provoke alteration of surface membrane polypeptides as revealed by iodination. Fibroblasts also triggered lymphocyte locomotion in serum-free medium, but their triggering effect was enhanced markedly by serum. The development and maintenance of lymphocyte motility required protein synthesis. These data suggest that during contact with fibroblasts lymphocytes acquire locomotor capacity by a mechanism different from activation to blast transformation.     

Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between beta-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105000 x g), isolated from the tumor cells was used as a source for actin quantification. The higher level of beta-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of beta-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. beta-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-beta actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) beta-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and beta-actin expression.     

Increased Antibiotic Sensitivity in Pseudomonas aeruginosa Following Passage in Carbenicillin-containing Media

Twelve dissimilar clinical isolates and 4 type cultures of Pseudomonas aeruginosa have been repeatedly passaged on agar containing 200 μg carbenicillin/ml. Passaged variants were compared with control organisms for their sensitivities to a range of antibiotics initially by a multodisk test and subsequently by serial dilution in agar. Two of the variants, both derived from clinical isolates, showed pronounced increases in sensitivity to several antibiotics, particularly kanamycin, neomycin, gentamicin and colistin sulphate. In some instances the minimum inhibitory concentration (MIC) for the passaged variants was 32–64 times lower than that for the control organisms. These potentiations contrast with previous results obtained by other workers for P. aeruginosa . In addition, several other of our passaged variants developed a more moderate degree of enhanced sensitivity to a limited number of antibiotics. Eight (67%) of the clinical isolates and one type culture did not become more sensitive to any of the antibiotics tested following carbenicillin passage. Onset of increased antibiotic-sensitivity varied with the strain, particular antibiotic and medium employed for passage. Although the addition of sucrose (0·5 M) and magnesium sulphate (0·01 M) to the passage medium appeared to delay development of antibiotic-sensitivity their presence eventually encouraged larger potentiations in antibiotic activity. The significance of the conversion of P. aeruginosa into forms with increased susceptibility to several antibiotics during chemotherapy with carbenicillin is discussed.     

Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones

We have analyzed the complexity of a unique tumor-specific transplantation antigen expressed by the murine ultraviolet light-induced fibrosarcoma 1591-RE. This tumor is highly immunogenic and is regularly rejected by normal mice. We have derived a cloned cytolytic T cell line showing a reactivity pattern representative of the cytolytic response of the host rejecting this regressor tumor. Using this T cell line (anti-A), variants of 1591-RE (1591-A-) were selected in vitro that had lost the same antigen as progressor variants of 1591-RE selected by the host in vivo. The in vitro derived variant was then used to generate a second T cell clone (anti-B) that recognized an antigen on the parental tumor that had been retained by the variants derived in vitro. Host-selected progressor variants were also found to have retained this antigen. By selecting for variants in vitro from the parental tumor with the anti-B T cell line, it was shown that the two different antigens (A and B) present on the parental tumor were lost independently of each other. Despite the independence of these two antigens, the host T cell response to the parental regressor tumor was invariably restricted to only the "immunodominant" A antigen.     

Clonal analysis of expression of tumor-associated transplantation antigens and of metastatic capacity

Summary ESb, a spontaneous high metastatic variant of the chemically induced T lymphoma Eb, was found previously to express a tumor-associated transplantation antigen (TATA) that was different from that of the parental line. Syngeneic tumor-specific cytolytic T lymphocytes (CTL) were able to recognize the different TATAs of Eb and ESb in vitro and could therefore be used for routine typing. The object of this study was to investigate tumor antigen expression on a clonal level and to compare the in vitro data with the in vivo behavior of the same cell lines.Our CTL typing analysis of cloned tumor lines revealed that the two populations, Eb and ESb, are distinct and relatively homogeneous with regard to their TATA expression. Furthermore, all ESb clones formed rosettes with antibody-coated erythrocytes, while none of the parental type Eb clones showed this characteristic. The sensitivity to tumor-specific CTL lysis varied with time of tumor cell culture in vitro in a clone-dependent manner.Variability was also noted in vivo in tumor growth and metastatic spread. Of over 50 ESb clones tested, the majority were highly metastatic while a minority were significantly lower in metastatic capacity. High and low metastatic ESb clones could not be distinguished by their expression of TATAs and of Fc receptors. There was also a considerable individual variability in the hosts, although they were genetically identical. This variability was most probably due to differences in the immune status of the animals.     

Trypanosoma evansi: Identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain

African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts’ antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.     

Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells.In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.Key words: actin, cofilin, ezrin, colon adenocarcinoma.     

Phenotypic diversity of cloned lines of Leishmania major promastigotes

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.     

Effect of changes in mineral composition and growth temperature on filament length and flagellation in the Archaeon Methanospirillum hungatei

Methanospirillum hungatei strains GP1 and JF1 when cultivated at 37°C in JMA medium grew as motile single cells or short chains of cells (typically 10–30 m long). When M. hungatei was grown in low Ca²⁺ concentrations or with the divalent cation chelator EDTA, the organism grew as long non-flagellated filaments (up to 900 m long). The two strains had different thresholds of calcium concentrations for long filament formation (<0.25 mM for GP1 and <0.15 mM for JF1) as well as different minimal Ca²⁺ requirements for growth. Both strains produced long, almost straight, filaments at Ca²⁺ concentrations near the minimum required for growth. At suboptimal growth temperatures the organisms still grew as short filaments but no longer possessed flagella. Western blot analysis indicated that flagellin monomer was present in cultures of long non-flagellated filaments and short non-flagellated cultures grown at suboptimal temperatures. The amount of flagellin present appeared to be equal in both non-flagellated and flagellated cultures. When cells were grown as long non-flagellated filaments and switched to growth conditions inducing short, flagellated forms, flagella were first observed at 2.5 h after this switch.Portions of this work were previously presented at the 91st General Meeting of the American Society for Microbiology, May 5–9 1991, Dallas, Texas (abstract I-81) and at the 41st Annual Meeting of the Canadian Society of Microbiologists, June 3–6 1991, London, Ontario (Abstract MP-1)     

Establishment and characterization of cell lines from the Walker carcinoma 256 able to grow in suspension culture and deficient in thymidine kinase

A cell line from the Walker carcinosarcoma 256 of the rat has been established in suspension culture in medium with 5% bovine calf serum for over 350 generations, with an average population doubling time of 17 h, a plating efficiency of 56%, a colony forming efficiency of 32%, and a good capacity to form colonies in soft agar. The cells are morphologically indistinguishable from those in the solid tumor and ascites as checked by transmission and scanning electron microscopy. The karyotype is characterized by a modal number of 65 chromosomes and by the presence of a marker metacentric chromosome. The cells express thymidine kinase, gamma-glutamyl transpeptidase, and alkaline phosphatase; are agglutinable by concanavalin A; and can be synchronized by the triple thymidine block. They induce primary tumors, both subcutaneously (solid) and intraperitoneally (ascitic), in the rat; are able to metastasize upon injection by the tail vein; and invade the chorioallantoic membrane of the chick embryo. Cells in suspension can be transferred to monolayers, considerably decreasing their tumorigenicity without affecting the other parameters studied, and can be switched back to suspension culture. DNA-mediated transfection showed that DNA from these cells can transform the NIH-3T3 line. Upon growth of the monolayers in a BrdUr-containing medium, a sub-line was established that was cloned into a thymidine kinase-deficient line unable to grow in HAT medium and with properties otherwise similar to those of the parental wild type cells.