Background and purpose:
Macrophages release cytokines that may contribute to pulmonary inflammation in conditions such as chronic obstructive pulmonary disease. Thus, inhibition of macrophage ...cytokine production may have therapeutic benefit. p38 MAPK may regulate cytokine production, therefore, the effect of two p38 MAPK inhibitors, SB239063 and SD‐282, on the release of TNF‐α, GM‐CSF and IL‐8 from human macrophages was investigated.
Experimental approach:
Cytokine release was measured by ELISA. Immunoblots and mRNA expression studies were performed to confirm p38 MAPK isoform expression and activity. Macrophages were isolated from lung tissue of current smokers, ex‐smokers and emphysema patients and exposed to lipopolysaccharide. These cells then released cytokines in a concentration‐dependent manner.
Key results:
SB239063 only inhibited TNF‐α release (EC50 0.3±0.1 μM). Disease status had no effect on the efficacy of SB239063. SD‐282 inhibited both TNF‐α and GM‐CSF release from macrophages (EC50 6.1±1.4 nM and 1.8±0.6 μM respectively) but had no effect on IL‐8 release. In contrast, both inhibitors suppressed cytokine production in monocytes.
Conclusions and Implications:
The differential effects of p38 MAPK inhibitors between macrophages and monocytes could not be explained by differences in p38 MAPK isoform expression or activity. However, the stability of TNF‐α mRNA was significantly increased in macrophages compared to monocytes. These data suggest a differential involvement for p38 MAPK in macrophage cytokine production compared with monocytes. These effects are not due to lack of p38 activation or p38α expression in macrophages but may reflect differential effects on the stability of cytokine mRNA.
British Journal of Pharmacology (2006) 149, 393–404. doi:10.1038/sj.bjp.0706885
Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the ...effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 μM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.
In this brief, a new form of direct conversion sigma-delta modulator-based receiver is presented. Distortion reduction is obtained as a result of including the down conversion mixers into the ...sigma-delta loop. The use of an equivalent low-pass model simplifies the analysis and design. The proposed architecture is very attractive for integration into any suitable CMOS technology. Simulation of a discrete equivalent model shows agreement with predicted performance.
The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic ...processing to generate a functional N- and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser super(397), which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3 beta (GSK-3 beta ) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser super(397) position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3 beta . Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF super(.)CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser super(397) by GSK-3 beta regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF super(.)CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.
We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including ...approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.
The majority of cases with early onset familial Alzheimer's disease have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 protein is a component of a high molecular weight ...membrane-bound complex that also contains β-catenin. The physiological relevance of the association between PS1 and β-catenin remains controversial. In this study, we report the identification and functional characterization of a highly conserved glycogen synthase kinase-3β consensus phosphorylation site within the hydrophilic loop domain of PS1. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, indicates that PS1 residues Ser353 and Ser357 are glycogen synthase kinase-3β targets. Substitution of one or both of these residues greatly reduces the ability of PS1 to associate with β-catenin. By disrupting this interaction, we demonstrate that the association between PS1 and β-catenin has no effect on Aβ peptide production, β-catenin stability, or cellular susceptibility to apoptosis. Significantly, in the absence of PS1/β-catenin association, we found no alteration in β-catenin signaling using induction of this pathway by exogenous expression of Wnt-1 or β-catenin and a Tcf/Lef transcriptional assay. These results argue against a pathologically relevant role for the association between PS1 and β-catenin in familial Alzheimer's disease.
The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic ...processing to generate a functional N- and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser(397), which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3beta (GSK-3beta) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser(397) position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3beta. Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF.CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser(397) by GSK-3beta regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF.CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.
The majority of cases with early onset familial Alzheimer's disease have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 protein is a component of a high molecular weight ...membrane-bound complex that also contains beta-catenin. The physiological relevance of the association between PS1 and beta-catenin remains controversial. In this study, we report the identification and functional characterization of a highly conserved glycogen synthase kinase-3beta consensus phosphorylation site within the hydrophilic loop domain of PS1. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, indicates that PS1 residues Ser(353) and Ser(357) are glycogen synthase kinase-3beta targets. Substitution of one or both of these residues greatly reduces the ability of PS1 to associate with beta-catenin. By disrupting this interaction, we demonstrate that the association between PS1 and beta-catenin has no effect on Abeta peptide production, beta-catenin stability, or cellular susceptibility to apoptosis. Significantly, in the absence of PS1/beta-catenin association, we found no alteration in beta-catenin signaling using induction of this pathway by exogenous expression of Wnt-1 or beta-catenin and a Tcf/Lef transcriptional assay. These results argue against a pathologically relevant role for the association between PS1 and beta-catenin in familial Alzheimer's disease.
Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared ...samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).
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