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R Lucas

Publications and source records attributed to R Lucas.

At least 91 records · Page 5Linked to original sources
PubMed1993

Transforming growth factor beta 1: NMR signal assignments of the recombinant protein expressed and isotopically enriched using Chinese hamster ovary cells.

The transforming growth factor beta s are a homologous family of multifunctional cytokines that regulate cell growth and differentiation. As a prelude to studies of the solution structure and dynamics of TGF-beta 1, we report virtually complete assignment of 1H and 15N resonances for this 25-kDa homodimeric protein. Recombinant TGF-beta 1 was expressed in Chinese hamster ovary cells. The cells were grown either in a completely 15N-enriched medium or in a medium containing selectively 13C, 15N-labeled amino acids to obtain either uniformly or specifically labeled protein, respectively. Two- and three-dimensional heteronuclear edited magnetic resonance spectra of the uniformly 15N-labeled protein and three samples selectively labeled with 13C and 15N yielded assignments for 96% of the backbone amide and C alpha protons and 87% of the side chain protons. To our knowledge, this is the first report of the use of an animal cell expression system to obtain extensive isotopic enrichment in order to sequentially assign a protein. The methodology described herein for the isotopic enrichment and resonance assignments of TGF-beta 1 should be generally applicable to other eukaryotic proteins expressed by animal cells.

Amino Acid Sequence↗
PubMed1993

Transforming growth factor beta 1: secondary structure as determined by heteronuclear magnetic resonance spectroscopy.

Virtually complete backbone NMR signal assignments have been reported for transforming growth factor beta 1 (TGF-beta 1) [Archer et al. (1993) Biochemistry (preceding paper in this issue)]. Herein we report the secondary structure of the protein in solution on the basis of these assignments and proton NOE's observed in a variety of 2D and 3D heteronuclear NMR spectra. Regular elements of secondary structure derived from the NOE data consist of (a) three helices spanning residues Y58-H68, F24-G29, and N5-F8 and (b) several pairs of two-stranded antiparallel beta-sheets. The longest two-stranded sheet runs from residue L83 to V106 with a type II reverse turn at G93-R94 and a chain twist at residue N103-M104. These elements of regular structure were confirmed by hydrogen exchange, chemical shift, and coupling constant data. With the exception of residues G46-S53, which exhibit relatively few and weak intraresidue NOE's, residues in the rest of the protein adopt an irregular but well-defined structure. All peptide bonds are trans except for a cis peptide bond between Glu35 and Pro36. The structural characteristics observed for TGF-beta 1 in solution generally agree closely with the recently derived crystal structures of TGF-beta 2 [Daopin et al. (1992) Science 257, 369-374; Schlunegger & Grütter (1992) Nature 358, 430-434]. Several noteworthy differences were observed that may be related to function.

Amino Acid Sequence↗
PubMed1993

Murine tumour necrosis factor plays a protective role during the initial phase of the experimental infection with Trypanosoma brucei brucei.

Soluble extracts from salivarian trypanosomes (Trypanosoma brucei brucei, T. evansi and T. congolense) were shown to be capable of inducing murine tumour necrosis factor (mTNF) secretion, both in vivo and in vitro, whereas the soluble extract of an intracellular trypanosome (T. cruzi) failed to do so. Furthermore, the role of mTNF during the initial phase of experimental infections with T. brucei was studied by treating infected mice with mTNF-inducing trypanosoma soluble extract and with neutralizing monoclonal anti-mTNF antibodies. Treatment of the infected animals with different doses of T. brucei soluble extract resulted in a lower first parasitaemia peak (low lysate dose) and in a longer survival time or in a nearly total inhibition of parasite development (high lysate dose). Cotreatment of the infected mice with both anti-mTNF antibodies and a high dose of soluble extract completely restored the parasite development in both trypanosusceptible C3H/He mice and trypanosubtolerant CBA Ca mice, indicating a protective role of mTNF during the parasitaemia. Collectively these results suggest a negative influence of mTNF on T. brucei development in vivo.

Animals↗
PubMed1992

Cytokine production after intravenous or peritoneal gram-negative bacterial challenge in mice. Comparative protective efficacy of antibodies to tumor necrosis factor-alpha and to lipopolysaccharide.

The production of TNF-alpha, IL-1, and IL-6 was measured in mice after bolus i.v. Escherichia coli O111 LPS injections and during bacteremia induced either by bolus i.v. or by i.p. challenges of live E. coli O111. High but transient TNF-alpha peaks were observed after bolus i.v. LPS or bacterial challenges. In contrast, the levels during lethal peritonitis increased progressively to values 50- to 100-fold lower than the peak values observed after i.v. injections, and remained sustained until death. Whereas after i.v. challenge with 1000 LD50 of LPS, anti-TNF-alpha antibody fully protected mice from death and reduced serum IL-1 and IL-6 levels, anti-TNF-alpha antibody did not improve the survival of mice nor reduced serum IL-1 and IL-6 levels after i.p. bacterial challenge. In contrast to anti-TNF-alpha antibodies, anti-LPS antibodies were protective in the peritonitis model. Protection was accompanied by a striking reduction of bacterial numbers and of TNF-alpha, IL-1, and IL-6 levels in the serum, but the levels of these cytokines were only marginally affected in the peritoneal lavage fluid. This latter observation demonstrates that the local peritoneal cytokines did not diffuse readily into the circulation, thus suggesting that at least part of the circulating cytokines are produced systemically. In conclusion, the striking differences between cytokine profiles as well as the divergent efficacy of anti-TNF-alpha antibody after i.v. bolus and after i.p. challenges suggest that TNF-alpha may not be as important in the pathogenesis of lethal peritonitis than after lethal acute bacteremia.

Animals↗
PubMed1992

Incidence of zidovudine-resistant human immunodeficiency virus isolated from patients before, during, and after therapy.

The zidovudine sensitivity of 372 isolates of human immunodeficiency virus (HIV) obtained from 237 patients before, during, and after treatment with zidovudine was examined. Virus resistant to > 0.5 micrograms/mL (1.87 microM) zidovudine was isolated from most patients (93%) after 36 months of therapy. Zidovudine-sensitive virus was isolated from 5 of 15 patients who had ended antiretroviral therapy but had previously shed resistant virus. The emergence of sensitive virus after end of therapy appeared to be influenced by both the duration of treatment and the time off drug. Patients with resistant virus tended to have low CD4 cell counts and HIV antigenemia at the commencement of therapy, suggesting that these two factors are important in the development of drug resistance.

CD4 Antigens↗
PubMed1992

Mechanisms underlying trypanosome-elicited immunosuppression.

T-cell proliferative responses of lymph node cells are profoundly suppressed during experimental infections of mice with Trypanosoma brucei. The active suppression of lymph node T-cell proliferative responses is attributed to the coexistence of at least two unlinked suppressive mechanisms that block different T-cell regulatory steps and operate through different effector mechanisms. The generation of prostaglandin-producing macrophages is entirely responsible for the suppression of IL-2 production whereas the induction of a prostaglandin-independent suppressive mechanism accounts for the suppression of the expression of IL-2 receptors (IL-2R). Both mechanisms are mediated by the cells that co-purify which macrophages. Despite an impairment at the level of T-cell proliferation, lymph node cells from T. brucei infected animals produce substantial amounts of interferon-gamma (IFN-gamma) and this lymphokine participates in the down-regulation of IL-2R expression. T-brucei-pulsed macrophage cell lines acquire concomitantly the potential to suppress T-cell proliferative responses and to stimulate CD8+ T-cells to secrete IFN-gamma. The sensibilization of CD8+ T cells by T. brucei-pulsed macrophages might be mediated by TNF-alpha. Collectively, these results indicate that the uptake of T. brucei by macrophages, either in vivo or in vitro, results in the generation of suppressive cells that annihilate T-cell proliferative responses. Furthermore, at least two cytokines (i.e., TNF-alpha and IFN-gamma) are released during these interactions. Besides playing a role in the pathway of T-cell immunosuppression, TNF-alpha and IFN-gamma could also contribute to immunopathological features that occur during trypanosome infections.

Animals↗
PubMed1991

Two discrete types of tumor necrosis factor-resistant cells derived from the same cell line.

From the murine fibrosarcoma cell line L929s, which is sensitive to tumor necrosis factor (TNF)-mediated cell lysis, two discrete types of TNF-resistant variants were derived by TNF selection. Cells of the first type (named L929r1) were not sensitized to TNF cytotoxicity by cotreatment with either inhibitors of protein or RNA synthesis, or gamma-interferon, despite the presence of a functional gamma-interferon response. L929r1 constitutively produced TNF in the supernatant and expressed membrane-bound TNF, which was not bound to the TNF receptor. In fact, TNF receptors could not be demonstrated on L929r1 cells, not even after low pH treatment and/or incubation with antiserum to TNF. L929r1 exhibited a stable TNF-resistant phenotype in the absence of further TNF selection. No evidence could be obtained that TNF acted as an autocrine growth factor for these cells. L929r2, the second type of TNF-resistant L929 cells, became sensitive to TNF lysis in the presence of RNA or protein synthesis inhibitors, or in the presence of gamma-interferon. TNF induced the secretion of interleukin 6 in these cells, additionally showing that functional TNF signaling in these cells indeed takes place, but does not lead to cell lysis under normal conditions. L929r2 did not produce TNF, also not upon stimulation with exogenous TNF. The number and binding affinity of TNF receptors were not consistently different between L929s and L929r2 cells. In the absence of further TNF selection, L929r2 gradually reverted to TNF sensitivity. This sensitivity was not reversible to TNF resistance by the gene-regulatory agents 5-azacytidine or sodium butyrate. Treatment with these agents also did not affect the TNF sensitivity of L929s cells nor the TNF resistance of L929r1 and L929r2 cells. In summary, our results suggest the existence among cells of the same cell line of discrete mechanisms for acquisition of resistance to TNF-mediated cell lysis.

Animals↗
PubMed1990

Quadruple-drug therapy for Mycobacterium avium-intracellulare bacteremia in AIDS patients.

The mycobacterial response was evaluated for patients with Mycobacterium avium-intracellulare complex (MAC) bacteremia treated with a quadruple regimen of rifabutin, clofazimine, isoniazid, and ethambutol. Mycobacteremia was cleared in 22 of 25 patients who received this regimen, and 18 patients experienced complete resolution of symptoms associated with MAC infection. All of the patients were immunodeficient, with a mean CD4 cell count at the time of diagnosis of MAC infection of 54.7 +/- 54.6 cells/microliters. All MAC isolates were resistant to clofazimine, isoniazid, and ethambutol. Sixteen of 24 isolates were resistant to rifabutin. Improved results of treatment may be related to the use of a higher dose of rifabutin (300-600 mg/day) compared with other studies (150-300 mg/day), earlier diagnosis and institution of treatment, and synergy between rifabutin and ethambutol. The quadruple regimen used in this study appears effective in clearing mycobacteremia and in ameliorating symptoms of infection.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Using the partner in the psychosocial treatment of schizophrenia: a multiple single case design.

This report describes the treatment of six couples where one partner had a long psychiatric history and a long-standing diagnosis of schizophrenia. Using a multiple single case design, the effects of providing the couples with information about schizophrenia and systematic family management (comprising problem solving and communication training) were assessed. The results showed that providing information alone had no effect on presenting symptoms but did improve the partners' perception of their ability to cope and solve problems. While a causal effect of treatment on improvement was not conclusively proven, the cycle of readmission and relapse was altered.

Adaptation, Psychological↗
PubMed1990

Generation and characterization of a neutralizing rat anti-rmTNF-alpha monoclonal antibody.

A rat anti-recombinant mouse tumour necrosis factor-alpha (rmTNF-alpha) monoclonal IgM antibody (1F3F3) with high specific binding activity for rmTNF-alpha was generated. The 1F3F3 monoclonal antibody (mAb) neutralizes the cytotoxic activity in vitro of rmTNF-alpha on L929 cells and inhibits the binding of radiolabelled rmTNF-alpha to its putative receptor on L929 cells. The 1F3F3 mAb binds to monomeric, dimeric and trimeric rmTNF-alpha and does not bind to reduced rmTNF-alpha, indicating that the recognized epitope is sensitive to denaturation. Using the 1F3F3 mAb as a capturing antibody and a biotinylated anti-rTNF-alpha as a detecting antibody, we have developed a sandwich ELISA that can specifically detect biologically active mTNF-alpha with a detection limit of 10 pg mTNF-alpha/well. This assay correlates well with the classical L929 cristal violet assay for the detection of bioactive rmTNF-alpha in biological fluids. The 1F3F3 mAb inhibits various in vitro biological activities of the rmTNF-alpha, such as the TNF-alpha-mediated tumoricidal activity of activated macrophages, the rmTNF-alpha-dependent stimulation of neutrophil degranulation and the growth-promoting effect of rmTNF-alpha. In vivo the 1F3F3 mAb inhibits lipopolysaccharide (LPS)-induced endotoxic shock. In conclusion, the 1F3F3 mAb is a useful tool to probe rmTNF-alpha activity both in vitro and in vivo.

Animals↗
PubMed1989

Transient production and secretion of human transforming growth factor TGF-beta 2.

Transient transfection of simian COS cells with a recombinant plasmid encoding the human transforming growth factor TGF-beta 2 precursor protein results in the production of a latent, biologically inactive protein. Upon acidification, recombinant TGF-beta 2 exhibits full biological activity, including inhibition of mink lung epithelial cell growth, stimulation of anchorage-independent growth of murine embryonic fibroblasts, and competition for TGF-beta receptor binding. Further analysis of conditioned media with antiserum to either a pro- [amino acid (aa) residues 1-220] or mature [aa 297-414] peptide of the TGF-beta 2 precursor suggests that TGF-beta 2, similar to TGF-beta 1 production in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol. 7 (1987) 3418-3427], is initially synthesized as a larger precursor protein which is proteolytically cleaved to yield the mature 112-aa transforming growth factor.

Animals↗