Inflammatory cytokines activate p38 MAPK to induce osteoprotegerin synthesis by MG-63 cells

Inflammatory bone diseases are characterized by the presence of pro-inflammatory cytokines that regulate bone turnover. Osteoprotegerin (OPG) is a soluble osteoblast-derived protein that influences bone resorption by inhibiting osteoclast differentiation and activation. In the present study, we demonstrate that interleukin-1beta and tumor necrosis factor alpha induce OPG mRNA production and OPG secretion by osteoblast-like MG-63 cells. Maximum induction of OPG secretion by either cytokine requires activation of the p38 mitogen activated protein kinase (MAPK) pathway but neither the p42/p44 (ERK) nor the c-Jun N-terminal MAPK pathways. Induction of OPG mRNA by either cytokine is also p38 MAPK dependent. Taken together, these data indicate that cytokine-induced OPG gene expression and protein secretion are differentially regulated by specific MAP kinase signal transduction pathways.     

Generation of human anti-MUC3 IgG antibodies after in vitro immunization of naive peripheral blood B-lymphocytes

It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies. In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens and could find applications in tumour targeting and immunotherapy. Received: 12 October 2000 / Accepted: 11 January 2001     

Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli

The maturation of [NiFe]-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the [NiFe]-metal centre has been assembled. The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated. Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged. Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue. Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids. A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2. The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension. The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase.     

Harmful effects of mustard bio-fumigants on entomopathogenic nematodes

Mustard (Brassica and Sinapis spp.) green manures tilled into the soil preceding potato crops act as bio-fumigants that are toxic to plant–parasitic nematodes, providing an alternative to synthetic soil fumigants. However, it is not known whether mustard green manures also kill beneficial entomopathogenic nematodes (EPNs) that contribute to the control of pest insects. We used sentinel insect prey (Galleria mellonella larvae) to measure EPN infectivity in Washington State (USA) potato fields that did or did not utilize mustard green manures. We found a trend toward lower rates of EPN infection in fields, where mustard green manures were applied, compared to those not receiving this cultural control method. In a series of bioassays we then tested whether the application of two mustard (Brassica juncea) cultivars, differing in glucosinolate levels, disrupted the abilities of a diverse group of EPN species to infect insect hosts. Mustard-exposure trials were conducted first in laboratory arenas where EPNs were exposed to mustard extracts suspended in water, and then in larger microcosms in the greenhouse where EPNs were exposed to green manure grown, chopped, and incorporated into field soil. In all trials we used G. mellonella larvae as hosts and included multiple EPN species in the genera Steinernema (Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, and Steinernema riobrave) and Heterorhabditis (Heterorhabditis bacteriophora, Heterorhabditis marelatus, and Heterorhabditis megidis). In the laboratory, EPN infection rates were lower in arenas receiving mustard extracts than the control (water), and lower still when EPNs were exposed to extracts from plants with high versus low glucosinolate levels. Results were nearly identical when mustard foliage was soil-incorporated into greenhouse microcosms, except that the negative effects of mustards on EPNs developed more slowly in soil. Significantly, in arenas of both types one EPN species, S. feltiae, appeared to be relatively unaffected by mustard exposure. Together, our results suggest that the use of mustard bio-fumigants for the control of plant–parasitic nematodes has the potential to interfere with the biocontrol of insect pests using EPNs. Thus, it may be difficult to combine these two approaches in integrated pest management programs.     

Synonymous mutations in the core gene are linked to unusual serological profile in hepatitis C virus infection

The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99-124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of anti-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection.     

Increased apoptotic activity on inflammatory human placentas in spontaneous abortions during the first and second trimester of gestation: a histochemical and immunohistochemical study

The aim of this study was to investigate the role of apoptotic markers on inflammatory human placentas from spontaneous abortions during the first and second trimester of gestation and compare them to those without inflammation. Paraffin-embedded specimens from 76 placentas were investigated by conventional histology and immunohistochemistry using monoclonal antibodies against M30, Caspase 3, Caspase 8 and Caspase 9, as well as the terminal deoxynucleotidyl tranferase-mediated deoxyuridine triphosphate nick end labeling method. A higher prevalence of expression of apoptotic markers (94.4%) was observed in placentas associated with chorioamnionitis in comparison with those without inflammation. Our observations confirm that apoptosis is strikingly prevalent in placentas diagnosed with histologic chorioamnionitis, while the inflammation induces cell death.