Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

Ligand-induced modification of a surface cAMP receptor of Dictyostelium discoideum does not require its occupancy

In Dictyostelium discoideum amoebae, cAMP-induced phosphorylation of the surface cAMP receptor is associated with a discrete transition in its electrophoretic mobility. The native and modified forms of the receptor are designated R and D (Mr = 40,000 and 43,000). The relationship of the number of receptors which are modified as a function of the receptors which bind cAMP was investigated. Modification was assessed by determining the amounts of R and D forms in Western blots which detect all receptors whether or not they are exposed on the surface. Cyclic AMP or the analog, adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS), induced a loss of cAMP-binding activity (down-regulation), which was not accompanied by a loss of the receptor protein. About 60% of the receptors do not bind cAMP in the absence of Ca2+ and are unmasked by 10 mM Ca2+. However, the fraction of receptors which are modified in response to cAMP is equal in the absence or presence of Ca2+. (Rp)-cAMPs induces down-regulation (50%) but not modification. Addition of cAMP, following down-regulation by (Rp)-cAMPS, causes all receptors to be modified. cAMP induces both down-regulation (80%) and modification. Modification is more readily reversed than down-regulation: 30 min after removal of cAMP, receptors remain down-regulated (57%) but are found in the R form. All receptors shift to the D form when cAMP is readded to the cells. These results indicate that exposed, as well as cryptic and down-regulated receptors, are modified in response to the cAMP stimulus.     

Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in $\text{e}\text{e}$ embryos, (2) $\text{e}\text{e}$ larvae from posthatching onward are darker than normal white larvae, and (3) fully grown $\text{e}\text{e}$ animals are sterile.Experiments reported here show that eyelessness in $\text{e}\text{e}$ embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on $\text{e}\text{e}$ blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either $\text{e}\text{e}$ or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation.Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye.The sterility of $\text{e}\text{e}$ larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to $\text{e}\text{e}$ recipients.The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to $\text{e}\text{e}$ recipients.     

Low-molecular-weight compounds with anticoagulant activity from the scorpion <Emphasis Type="Italic">Heterometrus laoticus</Emphasis> venom

Low-molecular-weight compounds with anticoagulant activity were isolated from the scorpion Heterometrus laoticus venom. The determination of the structure of the isolated compounds by nuclear magnetic resonance and mass spectrometry showed that one of the isolated compounds is adenosine, and the other two are dipeptides leucyl-tryptophan and isoleucyl-tryptophan. The anticoagulant properties of adenosine, which is an inhibitor of platelet aggregation, is well known, but its presence in scorpion venom is shown for the first time. The ability of leucyl-tryptophan and isoleucyl-tryptophan to slow down blood clotting and their presence in scorpion venom are also established for the first time.     

Microbialite response to an anthropogenic salinity gradient in Great Salt Lake,Utah

A railroad causeway across Great Salt Lake, Utah (GSL), has restricted water flow since its construction in 1959, resulting in a more saline North Arm (NA; 24%–31% salinity) and a less saline South Arm (SA; 11%–14% salinity). Here, we characterized microbial carbonates collected from the SA and the NA to evaluate the effect of increased salinity on community composition and abundance and to determine whether the communities present in the NA are still actively precipitating carbonate or if they are remnant features from prior to causeway construction. SSU rRNA gene abundances associated with the NA microbialite were three orders of magnitude lower than those associated with the SA microbialite, indicating that the latter community is more productive. SSU rRNA gene sequencing and functional gene microarray analyses indicated that SA and NA microbialite communities are distinct. In particular, abundant sequences affiliated with photoautotrophic taxa including cyanobacteria and diatoms that may drive carbonate precipitation and thus still actively form microbialites were identified in the SA microbialite; sequences affiliated with photoautotrophic taxa were in low abundance in the NA microbialite. SA and NA microbialites comprise smooth prismatic aragonite crystals. However, the SA microbialite also contained micritic aragonite, which can be formed as a result of biological activity. Collectively, these observations suggest that NA microbialites are likely to be remnant features from prior to causeway construction and indicate a strong decrease in the ability of NA microbialite communities to actively precipitate carbonate minerals. Moreover, the results suggest a role for cyanobacteria and diatoms in carbonate precipitation and microbialite formation in the SA of GSL.     

Antigen presentation by CD1d contributes to the amplification of Th2 responses to Schistosoma mansoni glycoconjugates in mice

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.     

Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles,peroxisomes, and mitochondrial-associated membrane

Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPAR alpha ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30-45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPAR alpha agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.     

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.