The Bradyrhizobium japonicum nolA gene encodes three functionally distinct proteins.
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Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B. japonicum. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B. japonicum nod genes. The expression of both NolA2 and NolA3 requires the presence of NolA1. NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578. (+info)
Inhibition of translation and cell growth by minigene expression.
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A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs. (+info)
Maturation, activation, and protection of dendritic cells induced by double-stranded RNA.
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The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses. (+info)
Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.
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A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed. (+info)
Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.
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Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells. (+info)
Regulation of inducible nitric oxide synthase expression in beta cells by environmental factors: heavy metals.
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The expression of inducible NO synthase (iNOS) in pancreatic islet beta cells modulates endocrine cell functions and, at very high levels of NO production causes beta-cell death. We tested the hypothesis that environmental factors such as heavy-metal salts modulate iNOS expression in beta cells. A rat beta-cell line (insulinoma RINm5F) was cultured in the presence of low-dose interleukin (IL)-1beta for suboptimal induction of iNOS. PbCl2 (0. 1-10 microM) dose-dependently increased NO (measured as nitrite) formation (P<0.001). In contrast, HgCl2 suppressed nitrite production (0.1-10 microM, P<0.05). Measurements of iNOS activity by determining citrulline levels confirmed the potentiating effect of PbCl2 (P<0.05). There was a narrow time window of heavy-metal actions, ranging from -24 h (Hg2+) or -3 h (Pb2+) to +2 h, relative to the addition of IL-1beta. By semi-quantitative reverse transcriptase-PCR, enhanced levels of iNOS mRNA were found in the presence of Pb2+ (P<0.05) and decreased levels in the presence of Hg2+. The amount of iNOS protein as determined by Western blotting was increased in the presence of Pb2+. We conclude that Pb2+ upregulates and Hg2+ suppresses iNOS gene expression at the level of transcription, probably by acting on the signalling pathway. These observations may have important implications for understanding pathological effects of environmental factors on endocrine organ functions. (+info)
Intestinal trefoil factor binds to intestinal epithelial cells and induces nitric oxide production: priming and enhancing effects of mucin.
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Intestinal trefoil factor (ITF or TFF3), NO and epithelium-associated mucin have important roles in sustaining mucosal integrity in the gastrointestinal tract. In the present study we examined ITF-binding molecules on IEC-18 cells (an intestinal epithelial cell line) with the use of flow cytometry and localized these molecules on the cell surface by confocal microscopy. Furthermore, we studied the interaction of mucin and ITF and their co-operative effect on NO production by the epithelium. Stimulation of cells with mucin (5 mg/ml) for 90 min resulted in a 5-fold increase in ITF binding. Treatment of IEC-18 cells with actinomycin D or cycloheximide attenuated mucin-enhanced ITF binding. Ligand blot analysis confirmed the induction of ITF-binding protein in IEC-18 cells by mucin. These results indicate that transcriptional and translational mechanisms are involved in the effect of mucin. Treatment with ITF overnight resulted in a low level of nitrite production by the cells, a 5-fold increase over control, in a concentration-dependent manner. ITF-induced NO production was attenuated by 1400W, a selective type II nitric oxide synthase (NOS2) inhibitor. By immunoblotting we found that NOS2 was up-regulated by ITF treatment. Priming IEC-18 cells with mucin for 90 min enhanced the effect of ITF on NO production, suggesting that the up-regulation of ITF-binding molecules by mucin might be physiologically relevant. Taken together, these observations indicate (1) that ITF-binding molecules that are up-regulated by mucin exist on the intestinal epithelial surface, and (2) that ITF modulates epithelial NO production via the NOS2 pathway, which is enhanced by mucin. (+info)
Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel.
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Capacitative Ca2+ entry (CCE) is Ca2+ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca2+ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca2+ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca2+-permeable cation channels activated by agonists that stimulate IP3 formation-very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca2+ entry channel. The role of Trp's in Ca2+ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp's appear to enhance this type of Ca2+ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca2+ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca2+ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated Ca2+ channels. (+info)