A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-alpha but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-kappaB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-alpha. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.     

Stereoselective reactions of (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene derivatives form 27-nor-3,20,23,26-tetrahydroxy-cholesten-22-ones and related bromo ketones

The previously reported analog of pregnenolone having a 3,4-dihydro-2H-pyran attached via a Cz.sbnd;C bond to the C-20 position (1), stereoselectively reacts with m-chloroperoxybenzoic acid in methanol at -5 degrees C. Acid-catalyzed hydrolysis of the isolated intermediates gives good yields of mostly a new 27-norcholesterol analog: (20R,23R)-3,20,23,26-tetrahydroxy-27-norcholest-5-en-22-one-3-acetate (2a, and a smaller amount of its 23S enantiomer 2b). Three different conditions of epoxidation and methanolysis followed by acid-catalyzed hydrolysis typically produce approximately 2:1 ratios of the 23R:23S diastereoisomers with a C-23 hydroxy group at the new asymmetric center. Bromine also reacts stereoselectively with (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene (4) giving mostly (20R,23R)-23-bromo-3,20,26-trihydroxy-27-norcholest-5-en-22-one (7a). Thus both major steroidal products 2a and 7a have the same C-23R configuration. Assignment of molecular structures and the absolute configurations to 1 and 2a were based on elemental analysis, mass spectra, nuclear magnetic resonance, FTIR infrared spectroscopic analysis and X-ray crystallography. Mechanisms are discussed for stereochemical selectivity during epoxidation and bromination of the 3,4-dihydro-2H-pyranyl ring in 1 and 4.     

Functional opioid pathways are necessary for hypocretin-1 (orexin-A)-induced feeding

We investigated the interaction of the orexigenic neuropeptide, hypocretin-1 (Hcrt-1, also known as orexin-A), with endogenous opioids (also orexigenic neuropeptides). Rats were injected with naltrexone (NTX, nonspecific opioid antagonist) i.p., i.c.v., in the lateral hypothalamus (LH), and in the accumbens shell (AcbSh), and naloxone methiodide (nonspecific opioid antagonist unable to cross the blood brain barrier) was injected i.p. Rats were then injected with Hcrt-1 in the LH. Food intake was measured for up to 4h thereafter. Rats were also pretreated with NTX in the LH, with Hcrt-1 injected in the AcbSh. NTX suppressed Hcrt-1-induced feeding only when injected i.p., i.c.v., and in the AcbSh. These studies reveal the necessity for functional central opioidergic pathways involving the AcbSh, but not the LH in Hcrt-1-induced feeding.     

A model of pollen-mediated gene flow for oilseed rape

The development of genetically modified (GM) crops has precipitated the need for risk assessment and regulation of pollen-mediated gene flow. In response to this need we present a mathematical model to predict the spatial distribution of outcrossing between progenitor populations of oilseed rape. The model combines the processes of pollen dispersal and pollination, resulting from wind and insect activity. It includes the effects of post-pollination reproductive processes by relating the number of progeny to both pollen deposition and competition at the stigma. Predictions compare well with a range of experimental results for different-sized GM source crops (i.e. 0.0064-0.8 ha) and non-GM target crops with different fertilities (i.e. self-fertile to 80% male-sterile). For these comparisons, we represent the variation caused by wind and insect exposure as a constrained set of random functions and limit the range of insect transport to typical plant-scale distances. In addition, the model is used to examine the relative sensitivity to the factors that determine gene flow. Target-crop fertility and source-crop size are shown to be more important than other factors, including background pollen and the natural range of insect activity. The concept of isolation distance to regulate gene flow is most effective for self-fertile target crops, but is ineffective for male-sterile target crops with low background pollen.     

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

Homology of primate DNA fragments for estrous-associated oviductal glycoprotein

In this study, the DNA fragments encoded for oviduct EGP were amplified by PCR method. Corresponding sequences from chimpanzee, orangutan and human were similarly amplified, but we failed to amplify the corresponding DNA sequences from: spider monkey, salmon and several different species of rodents, bandicoot, sheep and pig. Through analyzing the restriction enzyme map and the DNA sequence of the amplified fragments from primates, the data suggest that monkey and human DNA encoded for EGP are genetically more closely related to one another than each of them to other species.     

Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice

To begin to develop in vivo model systems for the assessment of the contributions of specific organic anion transporter (OAT) family members to detoxification, development, and disease, we carried out a targeted disruption of the murine organic anion transporter 3 (Oat3) gene. Surviving Oat3(-/-) animals appear healthy, are fertile, and do not exhibit any gross morphological tissue abnormalities. No Oat3 mRNA expression was detected in kidney, liver, or choroid plexus (CP) of Oat3(-/-) mice. A distinct phenotype manifested by a substantial loss of organic anion transport capacity in kidney and CP was identified. Uptake sensitive to inhibition by bromosulfophthalein or probenecid was observed for taurocholate, estrone sulfate, and para-aminohippurate in renal slices from wild-type mice, whereas in Oat3(-/-) animals transport of these substances was greatly reduced. No discernable differences in uptake were observed between hepatic slices from wild-type and Oat3(-/-) littermates, suggesting Oat3 does not play a major role in hepatic organic anion uptake. Cellular accumulation of fluorescein was reduced by approximately 75% in CP from Oat3(-/-) mice. However, capillary accumulation of fluorescein-methotrexate was unchanged, indicating the effects of Oat3 loss are restricted to the entry step and that Oat3 is localized to the apical membrane of CP. These data indicate a key role for Oat3 in systemic detoxification and in control of the organic anion distribution in cerebrospinal fluid.     

Risk assessment of GM plants: avoiding gridlock?

Cultivation of genetically modified crops is presently based largely on four crops containing few transgenes and grown in four countries. This will soon change and pose new challenges for risk assessment. A more structured approach that is as generic as possible is advocated to study consequences of gene flow. Hazards should be precisely defined and prioritized, with emphasis on quantifying elements of exposure. This requires coordinated effort between large, multidisciplinary research teams.