Search PubMed⌕ Search

Biomedical subjects

V Paetkau

Publications and source records attributed to V Paetkau.

At least 37 records · Page 2Linked to original sources
PubMed1988

Phorbol diester-inducible, cyclosporine-suppressible transcription from a novel promoter within the mouse mammary tumor virus env gene.

The mouse T-cell lymphoma cell line EL4.E1 constitutively synthesizes mouse mammary tumor virus (MMTV) transcripts encoding either the entire proviral genome or segments of it. In addition to these conventional mRNAs, however, an mRNA of about 1 kilobase accumulates after induction of these cells with phorbol myristate acetate (PMA). The accumulation of this transcript is strongly inhibited by the immunosuppressive agent cyclosporin A. Its pattern of induction by PMA and suppression by cyclosporin A is thus the same as seen for several lymphokine mRNAs in these cells, including interleukin-2 and granulocyte-macrophage colony-stimulating factor. The short MMTV transcript is the most abundant PMA-induced transcript in EL4.E1 cells, but was not found in a series of other leukocyte tumor cell lines. It is initiated from a novel promoter within the env gene, and a segment of 1,161 nucleotides is then spliced out. The major part of the transcript is a copy of the long terminal repeat (LTR) of MMTV. The MMTV proviral genomes in these cells, and the short transcript, contain a 491-nucleotide deletion in the LTR compared with the normal MMTV provirus. The resulting open reading frame could encode a protein of molecular weight 22,800, which is a likely candidate for an LTR-related protein with a similar molecular weight recently described in this system (J. Racevskis, J. Virol. 58:441-449, 1986).

Animals↗
PubMed1988

Cloning of cDNA for the bovine IL-2 receptor (bovine Tac antigen).

We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gt11 bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5' untranslated region, followed by a 825-bp coding region, and a 3' untranslated region that contain 1600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3' and 5' non-coding regions were not as homologous, apart from a specific site in the 5'-untranslated region that contained a 5'-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydrophobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylation in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein.

Amino Acid Sequence↗
PubMed1987

A serine protease (CCP1) is sequestered in the cytoplasmic granules of cytotoxic T lymphocytes.

Based upon the use of a new predictive algorithm, three peptides were synthesized that correspond to likely antigenic sites of the cytotoxic T cell-specific protease cytotoxic cell protein 1 (CCP1). Antibodies raised against these peptides, under reducing conditions, bound to a single protein of m.w. 29,000 found in two actively cytotoxic T cells. This protein was absent from a "cytotoxic" T cell line that had lost its cytolytic properties. By using immunocytologic techniques, the protein was localized within cytoplasmic granules. In addition, granules purified from the cytoplasm of cytotoxic T cells were shown, by Western blot analysis, to contain the protein. The molecule detected by the antibodies behaves, upon reduction, similarly to a diisopropylfluorophosphate-binding protein. Thus, here we provide evidence that CCP1, a protein whose expression correlates with cytotoxicity, is contained within organelles which have been implicated in killing. These findings strongly suggest that CCP1 plays a key role in T cell-mediated target cell lysis.

Cell Line↗
PubMed1987

The immunostimulatory effect of T cells and T cell lymphokines on murine fetally derived placental cells.

Evidence for maternal immune recognition of the fetus can be found during pregnancy, yet the conceptus remains unharmed. Indeed, in some cases immunizing the mother with cells sharing histocompatibility antigens with the fetus is beneficial to fetal survival. This could be due to the effect of maternally derived lymphokines on placental growth and function, according to the immunostimulation hypothesis. We demonstrate here that placental cells in culture proliferate upon the addition of T cell-derived lymphokines. The lymphokine activity has been separated from IL 2 and B cell growth factor, and copurified with IL 3 and granulocyte-macrophage colony-stimulating factor (CSF-GM). Recombinant CSF-GM and recombinant IL 3 showed a similar effect. The placental cells that proliferate in culture are of fetal origin and are characterized by strong adherence, phagocytosis, nonspecific esterase staining, and response to the macrophage-specific colony-stimulating factor CSF-1. In addition, treatment of pregnant females with anti-thymocyte serum as well as anti-Ly-2.1 monoclonal antibody, at gestational times before Ly-2 antigen appearance in the fetus, leads to a reduction of the proliferative and phagocytic capacity of day 12 placentae. These results clearly demonstrate that maternal T cells act upon fetally derived placental cells to improve their proliferative and phagocytic potential, and thus provide evidence for the immunostimulatory role of these cells during pregnancy.

Animals↗
PubMed1987

Induction, suppression and superinduction of lymphokine mRNA in T lymphocytes.

The expression of several lymphokine gene is characterized by a common pattern of induction, suppression and superinduction. This pattern was studied at the level of cellular mRNA in the mouse T-lymphoma cell line EL4, the human T-leukemia line Jurkat and in normal human peripheral blood lymphocytes. Lymphokine mRNA was induced by stimulating the cells with the phorbol diester PMA (TPA), with or without T-lymphocyte mitogens. The induction of Interleukin-2, Interferon gamma and the Colony Stimulating Factor for granulocytes and macrophages was suppressed by Cyclosporin A at moderate concns. Furthermore, these mRNAs accumulated to extraordinarily high levels (superinduction) if the protein synthesis inhibitor cycloheximide was added during transcription. Superinduction was not due to an increased rate of transcription. CsA interrupted ongoing transcription of IL2 by a mechanism not dependent on the induction of a new protein. The co-ordinate regulation of these genes strongly suggests that common intracellular signals mediate their expression.

Animals↗
PubMed1986

Random assignment of treatments in a 96-well (8 X 12) microtiter plate. A practical method.

A computer program, written in BASIC, has been developed to assign treatments strictly at random to the wells of a microtiter plate. A hard copy is printed of the treatment numbers with their assigned wells, followed by an 8 X 12 matrix of the treatment numbers. When a microtiter plate is placed over the matrix these numbers may be viewed directly through the wells. A program listing is reproduced. The effects of randomization on within-plate variation have been investigated and compared with two systematic methods of assignment.

Analysis of Variance↗
PubMed1986

Conversion of specific to nonspecific cytotoxic T lymphocytes.

The type of cytotoxic T lymphocyte (CTL) generated during a mixed leukocyte culture has been shown to depend on the purity of interleukin 2 (IL 2) used to stimulate growth. Antigen-specific CTL requiring both antigen and IL 2 to proliferate or express cytotoxicity (designated Type I in this work) are generated and maintained with highly purified IL 2. In the presence of IL 2 alone, they persist in a quiescent, noncytotoxic state, and on its removal they rapidly die. A second type of CTL (Type II), of nonspecific cytotoxicity, and dependent only on IL 2 for growth, is generated by exposure to an impure lymphokine preparation containing IL 2. Type I cells are T lymphocytes which are nongranular and nonadherent, whereas Type II cells are larger, adhere to plastic, and contain many granules. Cloned Type I cells are converted to Type II under three conditions: by low levels of IL 2 in the presence of a crude lymphokine preparation; by high levels of IL 2 in the presence of antigen; or by high levels of IL 2 in the presence of the phorbol diester PMA. Conversion results from an effect of the growth conditions on individual cells, and not from selection of a minor population.

Animals↗
PubMed1986

Translocation of murine interleukin 2 into microsomes during translation in a cell-free system.

Interleukin 2 (IL2) is a lymphokine which stimulates the growth of T lymphocytes. Although IL2 mRNA is translated into biologically active IL2 relatively efficiently in microinjected Xenopus laevis oocytes, it has been difficult to establish reproducible cell-free translation systems for this lymphokine. Such systems would be useful for the analysis of translational and post-translational events. In this paper, we show that a wheat germ extract will translate IL2 mRNA into biologically active murine IL2, most of which is translocated into dog pancreas microsomes when these are present. Translocation occurs only during translation, but wheat germ extracts translate IL2 mRNA whether microsomes are present or not. Surprisingly, reticulocyte lysates do not readily translate IL2 mRNA, and strongly inhibit its translation in wheat germ extracts. This inhibition can be partially alleviated by adding dog pancreas microsomes to the system. The inhibition seen with reticulocyte lysate may be attributable to the action of the signal recognition particle, which binds nascent secretory proteins, and blocks their further translation in the absence of the docking protein present in microsomes.

Animals↗
PubMed1986

Activation of cation transport by lymphokines in B cells without induction of DNA synthesis or immunoglobulin gene transcription.

We report on an experimental model that permitted us to evaluate the biologic relevance of membrane-associated biochemical events with respect to cell proliferation and maturation, each induced by distinct sets of signals. Antigen-affinity-enriched murine B cells cultured in the presence of a proliferative signal induced by LPS showed activation of Na+/K+ ATPase and enhanced the uptake of proline, followed by RNA, protein, and DNA synthesis, without the generation of antibody. Stimulation with both the proliferative signal(s) and the maturation signal(s) derived from lymphokines of an EL-4 thymoma induced B cells to proliferate and synthesize mRNA encoding mu-chain of IgM and to mature into IgM-secreting cells. Most important, the secretory product of EL-4, in the absence of LPS, activated Na+/K+ ATPase but failed to stimulate uptake of proline and synthesis of DNA or mu-specific mRNA. A similar response was observed in splenocytes depleted of T cells and in unfractionated spleen cells. Thus a component secreted by EL-4 can induce some of the early molecular events characteristic of the proliferative response but lacks the ability to initiate blast transformation and DNA synthesis.

Animals↗
PubMed1985

Is IL-2 regulated by a serum inhibitor?

Normal murine serum inhibits the proliferation of cloned cytotoxic T lymphocytes driven by pure interleukin 2 (IL-2), indicating that a component of normal murine serum is directly inhibitory to IL-2-dependent proliferation. However, the effect is not specific to such cells, since an IL-2-independent variant cell, and a number of lymphoid tumor cell lines are similarly inhibited. Addition of purified IL-2 does not overcome the inhibition, although its degree is reduced. Fractionation of murine serum showed that there are at least two inhibitory activities, which migrate with globular proteins of molecular weights greater than or equal to 10(6) and 4 X 10(4), respectively, on gel chromatography. Neither of the activities was specific for IL-2-dependent cells. Furthermore, murine IL-2 is stable in murine serum in vitro, although it disappears rapidly from the circulation after intravenous injection. It is therefore unlikely that serum inhibitor of IL-2 is an important immunoregulator in vivo.

Animals↗
PubMed1985

Molecular biology of interleukin 2.

Study of the lymphokine interleukin 2 (IL2) began with several diverse qualitative observations in the middle 1970s. Rationalization of early work led to a focused program of research involving a number of laboratories. This recently culminated in the molecular description of both murine and human IL2. The glycoprotein, containing about 16 000 daltons of protein, is secreted in response to activators of T helper lymphocytes. A number of complex immune responses is stimulated by pure, recombinant IL2. The induction of IL2 and other lymphokines is apparently specific, in that only a small subset of proteins shows altered expression in the response. Moreover, the immunosuppressive agent cyclosporin A specifically inhibits the induction of mRNA for IL2 and certain other lymphokines, without affecting the expression of the majority of other proteins. Understanding of IL2 and its biological context has recently been enhanced by an intense activity on the part of biotechnology companies.

Animals↗
PubMed1985

Abrogation of anti-Pichinde virus cytotoxic T cell memory by cyclophosphamide and restoration by coinfection or interleukin 2.

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.

Animals↗
PubMed1985

Biologic activities of recombinant human interleukin 2 on murine lymphocytes.

Recombinant human IL-2, produced by yeast cells, was tested in a number of in vitro responses with murine lymphocytes. The responses studied included proliferation of a cloned murine T lymphocyte line, generation of cytotoxic responses, recall of cytotoxic memory, and restoration of responses in spleen cells taken from cyclophosphamide-treated mice. In all cases, the recombinant human IL-2 had the same activity as purified murine IL-2. The recombinant material represents a source of IL-2 free of other lymphokines. The responses described in this work can therefore be ascribed to the direct effects of human IL-2, of known sequence, on the cells of interest.

Animals↗
PubMed1985

Interaction of interleukin 2 with cells: quantitative analysis of effects.

The response of T lymphocytes to interleukin 2 (IL 2) is accurately described by a four-parameter logistic function. Both data generated by a theoretical model of IL 2-driven proliferation and experimental data conformed to this function for all doses of IL 2. Assays measuring either the rate of DNA synthesis or cellular metabolism were well described. The variance of response was not constant but increased in a predictable way. Weighting was therefore included in deriving a nonlinear curve-fitting program. The effects on response of cell density, time, and the T lymphocyte line used were examined. Assays gave reproducible estimates of potency when test preparations were compared with a standard preparation, but not otherwise. A model for IL 2 proliferation was derived on the basis of the two-state model of the cell cycle, with cells leaving a quiescent state randomly and then traversing the other stages of the cell cycle in a determinate way.

Animals↗
PubMed1984

Induction of interleukin 2 messenger RNA inhibited by cyclosporin A.

Cyclosporin A blocked production of the lymphokine interleukin 2 by activated T lymphocytes. In a human and a murine cell line this inhibition reflected an absence of interleukin 2 messenger RNA. Under conditions in which these cells are normally stimulated to secrete high levels of interleukin 2, they failed to do so in the presence of cyclosporin A. In both cell lines this failure was accompanied by an absence of interleukin 2 messenger accumulation.

Animals↗
PubMed1984

A new rule for analyzing homologous coding sequences in DNA.

A new rule is proposed for detecting homology between DNA sequences coding for proteins. Simple coding considerations predict that if two DNA sequences are homologous because of a common ancestry, they should share sequence similarities primarily in the same translation phase, with their codons aligned. Similarities which are in phase are shown to be more frequent between related sequences than between unrelated or random ones. But similar segments which are out of translation phase are no more frequent between related than unrelated sequences. Duplication and concatenation of genetic elements, followed by gene duplication and random mutations would lead to the patterns observed. Similarities are examined between various immunologically important sequences, including immunoglobulins, MHC products, and the T lymphocyte antigen Thy 1.

Antigens, Surface↗