Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.     

High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention

CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.     

Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain

The severe acute respiratory syndrome (SARS) epidemic was caused by the spread of a previously unrecognized infectious agent, the SARS-associated coronavirus (SARS-CoV). Here we show that SARS-CoV could inhibit both virus- and interferon (IFN)-dependent signaling, two key steps of the antiviral response. We mapped a strong inhibitory activity to SARS-CoV nonstructural protein 1 (nsp1) and show that expression of nsp1 significantly inhibited the activation of all three virus-dependent signaling pathways. We show that expression of nsp1 significantly inhibited IFN-dependent signaling by decreasing the phosphorylation levels of STAT1 while having little effect on those of STAT2, JAK1, and TYK2. We engineered an attenuated mutant of nsp1 in SARS-CoV through reverse genetics, and the resulting mutant virus was viable and replicated as efficiently as wild-type virus in cells with a defective IFN response. However, mutant virus replication was strongly attenuated in cells with an intact IFN response. Thus, nsp1 is likely a virulence factor that contributes to pathogenicity by favoring SARS-CoV replication.     

Development of Ascarophis sp. (Nematoda: Cystidicolidae) to maturity in Gammarus deubeni (Amphipoda)

Experimentally transmitted Ascarophis sp. (Spirurida) developed to adult worms in the invertebrate host, Gammarus deubeni (Amphipoda), collected in the intertidal zone in Passamaquoddy Bay, New Brunswick, Canada. The morphological development and growth of larval stages is very similar to other cystidicolids, which are found as adults in fish. Unlike virtually all other Spirurida, which require a vertebrate definitive host, infective larvae of Ascarophis sp. migrate from the invertebrate host musculature into the hemocoel where they molt twice to become adults. Gravid females appear at 80 days and 69 days post-infection at 10-12 C and 18-20 C, respectively. While there is little evident host reaction to the parasite within the muscle tissue, within the hemocoel there is hemocytic reaction to shed nematode cuticles, released eggs, and sometimes the worm itself, including some melanization. The worms are morphologically similar to Ascarophis sp. from G. oceanicus in the Baltic and White seas and among Ascarophis species from fish is most similar to A. arctica. It is suggested that Ascarophis sp. no longer requires a vertebrate host and is transmitted between amphipods either through death and disintegration of infected amphipods and dispersal of the nematode eggs, or more likely through cannibalism or necrophagy.     

Mapping the low palmitate fap1 mutation and validation of its effects in soybean oil and agronomic traits in three soybean populations

Key message

fap 1 mutation is caused by a G174A change in GmKASIIIA that disrupts a donor splice site recognition and creates a GATCTG motif that enhanced its expression.

Abstract

Soybean oil with reduced palmitic acid content is desirable to reduce the health risks associated with consumption of this fatty acid. The objectives of this study were: to identify the genomic location of the reduced palmitate fap1 mutation, determine its molecular basis, estimate the amount of phenotypic variation in fatty acid composition explained by this locus, determine if there are epistatic interactions between the fap1 and fap _nc loci and, determine if the fap1 mutation has pleiotropic effects on seed yield, oil and protein content in three soybean populations. This study detected two major QTL for 16:0 content located in chromosome 5 (GmFATB1a, fap _nc) and chromosome 9 near BARCSOYSSR_09_1707 that explained, with their interaction, 66–94 % of the variation in 16:0 content in the three populations. Sequencing results of a putative candidate gene, GmKASIIIA, revealed a single unique polymorphism in the germplasm line C1726, which was predicted to disrupt the donor splice site recognition between exon one and intron one and produce a truncated KASIIIA protein. This G to A change also created the GATCTG motif that enhanced gene expression of the mutated GmKASIIIA gene. Lines homozygous for the GmKASIIIA mutation (fap1) had a significant reduction in 16:0, 18:0, and oil content; and an increase in unsaturated fatty acids content. There were significant epistatic interactions between GmKASIIIA (fap1) and fap _nc for 16:0 and oil contents, and seed yield in two populations. In conclusion, the fap1 phenotype is caused by a single unique SNP in the GmKASIIIA gene.     

Elution of low molecular weight solutes from viable cells of Saccharomyces bisporus

Previous work has shown that high molecular weight compounds were released from Saccharomyces bisporus by -mercaptoethanol, 2 M KCl, 0.5 M KCl and osmotic shock without affecting viability of the cells. In this current experiment, it was shown that low molecular weight compounds were also eluted when cells were treated in sequence with the same reagents. Alanine, glutamate, serine, an unidentified amino acid, glucose, glycerol, and arabitol were all eluted by each of the first three reagents. The osmotic shock eluate contained a larger number and quantity of amino acids than the first three eluates but, otherwise, the compounds in this eluate were the same. One hundred percent of the cellular glycerol and 65–70% of the total amounts of the other above mentioned solutes were released by the 4 eluting treatments. A hot water treatment was needed to extract the remainder of these solutes. The hot water extract also contained almost all the cellular proline. It was suggested that the elutable solutes are contained by cells in compartments (or vesicles) whose membranes are accessible to the eluting reagents without affecting the plasmalemma.     

Sciatin Is a Transferrin-Like Polypeptide

Abstract: Sciatin, an acidic glycoprotein from chicken sciatic nerve, has myotrophic effects on avian skeletal muscle cells in culture. As sciatin was found to have certain structural similarities to transferrin, we further investigated the physicochemical characteristics of sciatin in order to determine the relationship between these two proteins. Sciatin was found to be strikingly similar to ovotransferrin in amino acid composition. In addition, amino acid sequence analysis revealed that sciatin and ovotransferrin had identical amino-terminal sequences for at least the first 20 amino acid residues. Chicken ovotransferrin, but not human serum transferrin, cross-reacted with rabbit antisciatin antibodies upon rocket immunoelectrophoresis and double immunodiffusion in agar. In addition, in the presence of bicarbonate, sciatin bound approximately 2 mol ferrous iron/mol protein. Using the purification procedure developed for sciatin, we purified a protein from chicken serum that cross-reacted with antisciatin serum, migrated at a position identical to that of sciatin or ovotransferrin on two-dimensional gel electrophoresis, had an amino composition very similar to ovotransferrin and sciatin, and had myotrophic effects on cultured muscle cells. From these data, we conclude that sciatin is a growth-promoting polypeptide closely related in structure to transferrin.     

Review of reproductive and developmental toxicity studies with isopropanol

Published studies for reproductive and developmental toxicity conducted with isopropanol have been conducted by the inhalation and oral gavage routes of administration. Interpretation of the data from these studies has resulted in discussions regarding NOAELs and additional benchmark dose modeling publications. Unpublished reproductive and developmental toxicity studies administered in the drinking water were also conducted by BIBRA, and the results of those studies are presented here. In addition, all of the reproductive and developmental toxicity studies conducted with isopropanol are summarized and evaluated for concordance of effects and NOAELs. Endpoints of concern for regulatory agencies were decreases in male mating index and reductions in postnatal pup survival. Original study reports were evaluated and data collated to address these two endpoints, and the data summarized. Data are presented suggesting that there were technical problems in the study that implied a decrease in male mating index, and based on the results from the drinking water studies, the weight of evidence suggests that isopropanol does not affect male mating or fertility at dose levels of up to 1000 mg/kg/day. The weight of evidence suggests that isopropanol can cause decreases in postnatal pup survival following oral gavage administration of 1000-1200 mg/kg/day to the dams. The NOAEL for this endpoint with oral gavage administration was 700 mg/kg/day. Indications of maternal toxicity were also an important predictor for decreased postnatal survival. Decreased postnatal pup survival was also noted in the drinking water studies with isopropanol with a LOAEL of 2278 mg/kg/day and a NOAEL of 1947 mg/kg/day.