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Results for “Clone tracing”

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PubMed1999

Human tonsillar germinal center T cells are a diverse and widely disseminated population.

Germinal center (GC) T cells are a poorly investigated population with a unique helper-inducer memory phenotype. Murine GC T cells have been demonstrated to be oligoclonal and antigen specific. The aim of this study was to investigate the diversity of human tonsillar GC T cells. Immunohistochemistry with a panel of different TCRVbeta family antibodies and sequencing of TCRgamma gene rearrangements obtained from individual microdissected GC demonstrated local diversity of human tonsillar GC T cells. No evidence of local clonal dominance or of a local migratory pathway from the T cell zone to adjacent GC was seen. Primers specific to the junctional region of the TCRgamma gene of individual GC T cell clones were designed, and used to trace the dissemination of the clones. One was found to be concentrated locally. Both clones studied were present in both of the patient's tonsils, suggesting widespread dissemination. In addition, no evidence of somatic hypermutation was found in the TCRgamma gene sequences, or in expressed TCRalpha and beta gene sequences obtained by reverse transcriptase-PCR from isolated tonsillar GC.

Child↗

PubMed1996

The expression of the human chorionic gonadotropin beta subunit gene depends on negative control.

Transcription of the human chorionic gonadotropin (hCG) genes begins in the very early embryo stage and decreases or even disappears in nonplacental tissues. We have studied the regulation of hCG-beta genes by cell fusion and by the reverse transcription-polymerase chain reaction (RT-PCR). The choriocarcinoma cell lines, JAR and JEG-3, express high levels of the hCG-beta subunit while HeLa cells express extremely low levels of it. Most HeLa x JAR and HeLa x JEG-3 fusion clones expressed only a trace of the hCG-beta subunit mRNA, while JAR x JEG-3 fusion clones still expressed high levels of the hCG-beta subunit. Most transcripts of the hCG-beta subunit genes in JAR and JEG-3 came from the hCG-beta 5 subunit. Even the trace amount of hCG-beta transcripts from fusion clones came mainly from the beta-5 gene. The results suggest that the expression of the hCG-beta subunit genes depends on negative control. Probably when embryonic cells differentiate to form nonplacental tissues, specific inhibitors may appear and inhibit the expression of the hCG-beta subunit genes.

Cell Fusion↗

PubMed2006

HTPSELEX--a database of high-throughput SELEX libraries for transcription factor binding sites.

HTPSELEX is a public database providing access to primary and derived data from high-throughput SELEX experiments aimed at characterizing the binding specificity of transcription factors. The resource is primarily intended to serve computational biologists interested in building models of transcription factor binding sites from large sets of binding sequences. The guiding principle is to make available all information that is relevant for this purpose. For each experiment, we try to provide accurate information about the protein material used, details of the wet lab protocol, an archive of sequencing trace files, assembled clone sequences (concatemers) and complete sets of in vitro selected protein-binding tags. In addition, we offer in-house derived binding sites models. HTPSELEX also offers reasonably large SELEX libraries obtained with conventional low-throughput protocols. The FTP site contains the trace archives and database flatfiles. The web server offers user-friendly interfaces for viewing individual entries and quality-controlled download of SELEX sequence libraries according to a user-defined sequencing quality threshold. HTPSELEX is available from ftp://ftp.isrec.isb-sib.ch/pub/databases/htpselex/ and http://www.isrec.isb-sib.ch/htpselex.

Binding Sites↗

PubMed1996

The identification of a novel cDNA preferentially expressed in the olfactory-limbic system of the adult rat.

To analyze cell-specific brain gene expression, we have developed a PCR-based subtractive hybridization cloning method utilizing trace starting material, allowing isolation of novel genes expressed under specific conditions. Our previous studies indicated that local substantia nigra (SN) type 1 astrocytes elaborate an array of trophic molecules which support the survival of SN dopaminergic neurons. Therefore, the current study focused on astrocyte gene expression utilizing a type 1 astrocyte-enriched cDNA library. We report initial characterization of a novel cDNA, designated AT1-46, that is preferentially expressed in the olfactory-limbic system of the adult rat brain. Although AT1-46 is expressed widely in the periphery, it is regulated both developmentally and in a cell-specific fashion in the brain. Structurally, AT1-46 is predicted to encode a highly alpha-helical molecule with several domains of potential coiled coil formation, and exhibits a 28% amino acid sequence identity with the intermediate filament-associated protein, trichohyalin.

Amino Acid Sequence↗

PubMed1978

Distribution of immunoglobulin-containing cells in bone marrow and lymphoid tissues in patients with monoclonal gammapathy.

Cell suspensions of bone marrow and lymphoid tissue from 85 patients with monoclonal gammapathy were investigated by a direct immunofluorescence procedure for the detection of intracellular immunoglobulin alpha, mu, gamma, kappa and lambda chains. Serum Ig levels were determined and daily syntheric rates estimated. In all cases the majority of Ig-containing bone marrow cells contained the same Ig class as that of the M-component in serum or urine indicating a diffuse distriubtion of these clones in the bone marrow. This was observed not only in myeloma but also in benign monoclonal gammapathy (BMG) and lymphoma with an M-component. The M-component producing clone could be traced to extramedullary lymphoid tissue in myeloma but usually not in BMG. A positive correlation was found between the calculated synthetic rate of the M-component and the number of Ig-containing cells in the bone marrow and some indication was found that the synthetic rate per cell might be lower in IgM and IgG than in IgA monoclonal gammapathy. The depressed level of polyclonal Ig in myeloma and to some extent in BMG was parallelled by a diminished number of Ig-containing cells in the bone marrow.

Adult↗

PubMed1983

Glycosphingolipids in clonal variants of rat fibrosarcoma cells with different transplantability.

Neutral glycolipids and gangliosides of four clonal variants of rat fibrosarcoma AS-653 cells with different transplantability were analyzed. A highly malignant clone A had a much lower quantity of GM3 ganglioside and a much higher quantity of lactosylceramide as compared to clones Z and G, which showed low transplantability and less malignancy, and a variant clone P, which showed tumorigenicity only in ascites form. A similar correlation was found with the quantity of an unidentified slow-migrating neutral glycolipid in various clones. This glycolipid was present in trace amount in the original highly malignant clone A, increased moderately in clones G and Z, and increased greatly in clone P, which showed no subcutaneous transplantability. The results of these studies suggest that a blockage of synthesis of GM3 ganglioside and a long chain neutral glycolipid occurred with enhanced malignancy, and an enhanced synthesis of GM3 ganglioside and the long chain neutral glycolipid is associated with a decrease in transplantability.

Animals↗

PubMed1998

Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction.

Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd' of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd' suggests that the inversion may be the result of the transposable element's activity. Characteristics of Odysseus' terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

Animals↗

PubMed1975

In vitro growth of neoplastic rat glial cells.

An ependymoma was induced in the brain of a BD-IX rat by repeated doses of MNU. In vitro grown cells of the tumor were fibroblasts and stellate cells considered to be glial. Reimplantation of these cells into the brain of another BD-IX rat resulted a tumor growing in the meninges. This tumor was then repeatedly transplanted over several passages. Its gross morphology resembled that of a sarcoma; however, glial in cells could be demonstrated with silver methods. A cell line derived from the first transplanted tumor group proved to contain S-100 protein in traces in the first passages in vitro. Explanted cells of the primary ependymoma were cloned. Two clones were established, RGL 1 and RGL 2. The cells of both clones resembled morphologically the stellate elements of the primary explant. RGL 1 contained S-100 protein. It was cloned again in the 16th subpassage. Four clones were established. Only 1 contained S-100 protein in traces. The clone RGL 1 had a subdiploid karyotype with a modal number of 39 and a large submetacentric marker. It was subcloned in the 25th passage with two clones, both having a karyotype of 41 chromosomes.

Animals↗

PubMed1992

Staphylococcal enterotoxin B as a potent suppressant of T lymphocytes: trace levels suppress T lymphocyte proliferative responses.

Staphylococcal enterotoxins have long been known to be powerful stimulators of T lymphocytes in mouse and man. In a previous study we showed that high concentrations of staphylococcal enterotoxin serotype B (SEB) failed to stimulate strong proliferative responses by Lewis rat T lymphocytes. Moreover, concentrations of SEB (10-50 micrograms/ml) that stimulated optimal mouse T lymphocyte proliferative responses suppressed a mitogen- or antigen-induced rat T lymphocytes proliferative responses. The present study shows that SEB at low concentrations (as low as 10(-3)-10(-4) micrograms/ml) and often also trace levels (about 10(-6)-10(-7) micrograms/ml) suppresses both rat and mouse T lymphocytes proliferative responses to mitogen or antigen. Furthermore, under different circumstances, SEB may have conflicting effects on the same T cells. While high concentrations (1-50 micrograms/ml) of SEB stimulate certain mouse T cell clones, low concentrations or trace levels have a potent suppressive effect on the same clones. The results indicate that the in vitro conflicting effects of SEB on the same T cells are concentration dependent and may reflect its in vivo effects on SEB-reactive T lymphocytes. The suppression of the mitogen- or antigen-induced stimulation of T cell clones by SEB was direct and did not require the agency of suppressor cells. Furthermore, the suppression by low amounts of SEB was not major histocompatibility complex restricted and affected a large proportion of both rat and mouse T lymphocyte subpopulation, regardless of their antigenic specificity. The concomitant suppressogenic and stimulatory characteristics of SEB support the conclusion that, under different conditions, SEB can be considered a "super-suppressogen" as well as a "super-antigen". Overall, the results suggest that SEB, and possibly other bacterial toxins, could be useful in immunomodulation of specific T cell responses.

Animals↗

PubMed1995

Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)-branch of diantennary oligosaccharides from secreted recombinant beta-trace protein.

The human beta-trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK-21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 which contain the cDNAs encoding the human secretory glycoproteins beta-trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chromatography and N-linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural beta-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of beta-trace protein coexpressed with human ST6N were found to contain NeuAc in alpha 2,6- or alpha 2,3-linkage in the same ratio. From 1H-NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both alpha 2,3- and alpha 2,6-linkage to Gal beta(1-4)GlcNAc-R, whereas 40% of the molecules of this fraction contained NeuAc in only alpha 2,3-linkage to Gal(beta 1-4)GlcNAc-R. The alpha 2,6-linked NeuAc was shown to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.

Animals↗

PubMed1989

The difficulty of cloning Streptococcus pneumoniae mal and ami loci in Escherichia coli: toxicity of malX and amiA gene products.

Stability problems are frequently encountered when cloning pneumococcal DNA in Escherichia coli multicopy plasmid vectors such as derivatives of ColE1. In this paper, we report our investigations of these problems using the pneumococcal mal and ami regions. We offer evidence that, in both cases, promoters are not the primary cause of cloning problems. Indeed, successful cloning of mal and ami promoters has been achieved with standard vectors (devoid of transcriptional terminators flanking the insertion site). Moreover, we show that the entire mal fragment can be introduced into an E. coli strain harboring a chromosomal mutation that reduces plasmid copy number. The cause of the cloning problems has been traced to the malX and amiA structural genes. Overexpression of these genes, which probably encode lipoproteins, could have deleterious effects on E. coli hosts, possibly as a result of impairing the protein export machinery.

Cloning, Molecular↗

PubMed2006

Isolation of immunoresistant human glioma cell clones after selection with alloreactive cytotoxic T lymphocytes: cytogenetic and molecular cytogenetic characterization.

Intratumoral heterogeneity and genetic instability within gliomas may allow intrinsically immunoresistant (IR) cells to escape alloreactive cytotoxic T lymphocyte (aCTL) cellular immunotherapy. The potential existence of aCTL-resistant variants prompted us to investigate whether cellular immunotherapy resistant glioma models could be isolated. To generate the models, repeated intermittent or continuous selective pressure (ISP or CSP) with multiple aCTL populations was applied to a low-passage glioblastoma cell explant, 13-06-MG, obtained from a patient at initial diagnosis. IL-6 and IL-8 secretion was greater in coincubates of aCTL cells with 13-06-ISP and 13-06-CSP immunoselected cells than those with 13-06-MG parental cells. Initially, the immunoselected cells were less sensitive to aCTL lysis; however, the reduced aCTL-sensitivity was not maintained upon further selection. We therefore isolated IR clones from continuously immunoselected cells (13-06-CSP). The frequency of IR clones was 1-6 cells per 10,000 immunoselected cells. Two clones selected for further study, 13-06-IR29 and 13-06-IR30, resisted aCTL lysis in the absence of immunoselective pressure. Cytogenetic analyses revealed structural anomalies and genomic imbalances unique to the IR clones. Based on these findings, a hypothetical model is proposed that traces the origin of the IR clones to a clonal variant within the 13-06-CSP and 13-06-MG populations.

Brain Neoplasms↗

PubMed2005

Primate trace amine receptor 1 modulation by the dopamine transporter.

Recently identified trace amine receptors are potential direct targets for drugs of abuse, including amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). We cloned full-length rhesus monkey trace amine receptor 1 (rhTA(1)) that was 96% homologous to human TA(1). The trace amines tyramine and beta-phenylethylamine (PEA) and the monoamine transporter substrates (+/-)-amphetamine and (+/-)-MDMA stimulated cAMP accumulation in rhTA(1)-expressing cell lines, as measured by a cAMP response element-luciferase assay. Cocaine did not stimulate cAMP accumulation in rhTA(1) cells, but it blocked [(3)H]PEA transport mediated by the dopamine transporter. Cotransfection with the human dopamine transporter enhanced PEA-, amphetamine-, and MDMA-mediated rhTA(1) receptor activation, but it diminished tyramine activation of rhTA(1). Because TA(1) (EGFP-rhTA(1) chimera) was largely intracellular, conceivably the dopamine transporter can facilitate access of specific agonists to intracellular TA(1). rhTA(1) mRNA expression was detected in rhesus monkey substantia nigra, implying that TA(1) may be colocalized with the dopamine transporter in dopamine neurons. In summary, primate TA(1) receptors are direct targets of trace amines, amphetamine, and MDMA. These receptors could also be indirect targets of amphetamine, MDMA, and cocaine through modification of monoamine transporter function. Conceivably, rhTA(1) receptors may be located on pre- or postsynaptic membranes. Interference with the carrier function of monoamine transporters with a consequent rise of extracellular levels of trace amines could activate these receptors. The cloning of a highly homologous TA(1) from rhesus monkey and demonstration that rhTA(1) receptors are activated by drugs of abuse, indicate that nonhuman primates may serve to model physiological and pharmacological TA(1)-mediated responses in humans.

Amino Acid Sequence↗

PubMed2002

Stimulation of the catecholaminergic and serotoninergic neurons in the rat brain by R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane, (-)-BPAP.

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane HCl, (-)-BPAP, the recently developed selective and much more potent catecholaminergic/serotoninergic enhancer (CAE/SAE) substance than (-)-deprenyl enhances the performance of midbrain neurons, both in vivo and ex vivo, in a characteristic complex manner, presenting one bell shape dose/concentration effect curve in the low nanomolar range and another at higher micromolar range. For example, 4.7 +/- 0.10 nmol/g wet weight noradrenaline was released within 20 min from the quickly removed locus coeruleus of saline treated rats. This amount was increased 30 min after the subcutaneous administration of 0.0005 mg/kg (-)-BPAP to 15.4 +/- 0.55 nmol/g (P < 0.001). However, following the injection of a hundred times higher, 0.05 mg/kg, dose of (-)-BPAP, the amount of noradrenaline (4.3 +/- 0.25 nmol/g) released from the locus coeruleus did not differ from the control value. In ex vivo experiments, when the isolated locus coeruleus was soaked in an organ bath containing (-)-BPAP, the release of noradrenaline was significantly enhanced from 10(-16) M concentration, reached a peak effect at 10(-13) M concentration, but 10(-10) M (-)-BPAP was ineffective. A significant enhancer effect was detected also in the high concentration range from 10(-8) M, the peak effect was reached at 10(-6) M concentration and 10(-5) M (-)-BPAP was ineffective. (-)-BPAP enhanced in the low concentration range the performance of dopaminergic and serotoninergic neurons with a peak effect at 10(-13) and 10(-12) M concentration, respectively. The results with (-)-BPAP, the highly specific artificial enhancer substance, suggest that (i) high and low affinity "enhancer" receptors may exist in the brain, and (ii) that they may be identified with the recently cloned family of the "trace amine" receptors, activated by beta-phenylethylamine and tryptamine, the prototypes of the endogenous enhancer substances.

Animals↗

PubMed2000

Critical role of enhanced CD4 affinity in laboratory adaptation of human immunodeficiency virus type 1.

Strains of human immunodeficiency virus type 1 (HIV-1) that use the coreceptor CXCR4 (X4 strains) become laboratory adapted (LA) when selected for ability to replicate in leukemic T cell lines such as H9. Compared with patient X4 viruses, the gp120-gp41 complexes of LA viruses have a constellation of common properties including enhanced affinities for CD4, greater sensitivities to inactivations by diverse antibodies and by soluble CD4, increased shedding of gp120, and improved abilities to infect HeLa-CD4 cell clones that contain only trace quantities of CD4. These common characteristics, which may result from a concerted structural rearrangement of the gp120-gp41 complexes, have made it difficult to identify a specific feature that is critical for laboratory adaptation. To test the hypothesis that replication of patient X4 HIV-1 is limited by the low CD4 concentration in H9 cells (7.0 x 10(3) CD4/cell), we constructed H9 derivatives that express at least 10 times more of this receptor. Interestingly, most patient X4 isolates readily grew in these derivative cells, and the resulting virus preparations retained the characteristics of primary viruses throughout multiple passages. In contrast, selection of the same viruses in the parental H9 cells resulted in outgrowth of LA derivatives. We conclude that a weak interaction of patient X4 HIV-1 isolates with CD4 is the primary factor that limits their replication in leukemic T cell lines.

Adaptation, Physiological↗

PubMed2002

Rational design of 'water-soluble' bacteriorhodopsin variants.

We have explored the interchangeability of soluble and membrane proteins by attempting to render a helical membrane protein 'water soluble' through mutation of its lipid-exposed residues. Using an atomic resolution structure of bacteriorhodopsin (bR), two different strategies were developed to identify lipid-exposed residues for mutation. In the first strategy all residues in trimeric bR with solvent accessibility >35% were marked for replacement. Replacement residues were chosen so as to map an average surface of helical soluble proteins onto the bR surface, resulting in the mutagenesis of 14.9% of surface residues. The second strategy took into account the observation that accessible residues can be categorized as fully or partially accessible. Consequently, three mutants were designed based on monomeric bR, all with their accessible residues changed and with varying extents of mutagenesis of partially accessible residues. 13.5-24.3% of the wild-type surface was altered in these designs. The construct for the first design was cloned into Escherichia coli. Trace amounts of the mutant protein were expressed with the concurrent overexpression of an endogenous prolyl isomerase. In contrast, all three mutant proteins of the second design expressed well and could be purified to homogeneity. Systematic refolding trials were undertaken with limited success at solubilization in aqueous media. We have discussed the feasibility of applying the 'solubilization strategy' outlined here to membrane proteins.

Amino Acid Sequence↗

PubMed1995

[Alfa-2a interferon induces molecular remission in post-BMT relapse of chronic myelogenous leukaemia. Report of a case with loss of bcr-abl RNA].

Alpha-interferon (alpha-IFN) has been used in relapsed CML post-BMT, cytogenetic responses being attained in a number of cases (33 to 42%). In first chronic phase-CML patients such cytogenetic response has been correlated with the disappearance of the bcr region rearrangement, as seen with Southern-blot, but when RT-PCR is used only a small number of patients maintain undetectable traces of the Ph1 clone. A case of CML in haematological and cytogenetic relapse after BMT is reported who showed criteria of "accelerated" phase and, after treatment with alpha-IFN achieved haematologic, cytogenetic and molecular remission (Southern-blot and PCR negative) and disappearance of the abnormal clone with recovery of the donor haemopoiesis. The duration of the alpha-IFN cytogenetic response is longer than that of BMT (5 vs 3.5 yr), which is noteworthy. Taking the low toxicity of alpha-IFN into account, as compared with that of the other choices (a second BMT, IL2), this treatment should be offered to all patients with cytogenetic relapse after BMT.

Antiviral Agents↗

PubMed2000

Spectral karyotyping of Werner syndrome fibroblast cultures.

Fibroblast cultures from two Werner syndrome patients were analyzed by spectral karyotyping. There were multiple, pseudodiploid clones in both cultures, mostly marked by random balanced reciprocal translocations. One of the cultures contained a clone with three-way exchanges involving chromosomes 2, 3, and 16. Duplication-deficiencies were exceptional, as were completely normal metaphases. The most frequent breakpoint occurred at 16q22 which corresponds to FRA16B, possibly reflecting difficulties of WS cells in replicating AT-rich repetitive DNA structures. Both cultures ceased proliferation after eight in vitro passages, but a single clone with exceptional growth potential emerged in one of the senescing cultures. Due to its identical translocations, the derivation of this near tetraploid clone (with tetrasomy for all autosomes except chromosomes 4 and 6) could be traced to the most prevalent pseudodiploid clone of the parental mass culture. Our study confirms the existence of variegated translocation mosaicism as the cytogenetic hallmark of WS fibroblast cultures and suggests that tetraploidization in combination with certain chromosome rearrangements and selective chromosome dosage may overcome the severely limited in vitro lifespan of WS fibroblasts.

AT Rich Sequence↗