Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1β. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.     

Sex pheromone of Argyrotaenia pomililiana (Lepidoptera: Tortricidae), a leafroller pest of apples in Argentina

Sex pheromone gland extracts of Argyrotaenia pomililiana Trematerra & Brown females contained seven 14-chain compounds, the Z and E isomers of 11-tetradecenyl acetate, 11-tetradecen-1-ol, and 11-tetradecenal, respectively, together with tetradecyl acetate. In field trapping tests, a 100:5 blend of Z11-14:Ac and Z11-14:Al was shown to be suitable for detection and monitoring of A. pomililiana. This species-specific lure will facilitate the use of mating disruption against codling moth, Cydia pomonella (L.), in Argentine fruit orchards.     

The pituitary response to ovine corticotropin-releasing hormone is enhanced in obese men and correlates with insulin resistance.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in central obesity has been demonstrated in women. We studied the corticotropin (ACTH) and cortisol response to ovine corticotropin releasing hormone (oCRH) and its association to parameters of adiposity and insulin resistance in a group of 19 healthy obese (BMI > 25 kg/m2) and 9 non-obese men. Relative insulin resistance was assessed by the homeostatic model assessment (HOMA IR). Baseline ACTH was similar, while cortisol was lower in the obese group. The ACTH response to oCRH was significantly higher in the obese group. ACTH incremental area under the curve (iAUC) correlated with age, HOMA IR, and sagittal diameter but not with leptin. In multiple regression analysis, only HOMA IR was an independent predictor of ACTH iAUC. In conclusion, obese men have hyperactivity of the HPA axis at the pituitary level, which appears to be linked to insulin resistance.     

Estradiol regulates susceptibility following primary exposure to genital herpes simplex virus type 2, while progesterone induces inflammation

We report here that sex hormones modulate susceptibility to a sexually transmitted viral agent, herpes simplex virus type 2 (HSV-2), in a mouse model. Ovariectomized mice were administered either saline (control), estradiol (E(2)), progesterone (P(4)), or a combination of both estradiol and progesterone (E+P) and infected intravaginally with HSV-2. With an inoculation dose of 10(5) PFU, the saline- and P(4)-treated mice were found to be highly susceptible to genital HSV-2 infection. Both groups had extensive pathology and high viral titers in vaginal secretions, and 100% of mice succumbed by day 4 postinfection. E(2)-treated mice were protected from HSV-2 infection at the same dose and did not display any vaginal pathology or viral shedding. There was a slow progression of genital pathology in the combination hormone-treated group, along with prolonged viral shedding; 80% of animals succumbed by day 13. With lower inoculation doses of 10(3) and 10(2) PFU, 50 and 100%, respectively, of the combination hormone-treated mice survived. Localization of HSV-2 infection showed extensive infection in the vaginal epithelium of P(4)- and saline-treated animals within 24 h of inoculation. E(2)-treated animals were clear of infection, while the E+P-treated group had focal infection at 24 h that had progressed extensively by day 3. Infection was accompanied by persistent inflammation and infiltration of neutrophils in the P(4)-treated group. An analysis of the genes in the vaginal tissue showed that inflammation in the P(4)-treated group correlated with local induction of chemokines and chemokine receptors that were absent in the E(2)-treated mice and in uninfected P(4)-treated mice. The results show that sex hormones regulate initiation of infection and immune responses to genital HSV-2 infection.     

Immunogold Localization of the L3 Protein of Maize Lipid Bodies during Germination and Seedling Growth

We have used antibodies directed against the 16.5 kilodalton protein L3, the most abundant integral protein of maize (Zea mays L. cv Mo 17) lipid bodies, to follow the fate of this protein in scutellar parenchyma cells of maize during germination and subsequent seedling growth. Using gel electrophoresis and immunoblotting as well as immunocytochemical electron microscopy, we found that the amount of L3 decreases gradually during the first 3 to 4 days of seedling growth and more rapidly over the course of the next several days. Immunogold localization of the protein on thin sections indicated that L3 is found exclusively in the surface phospholipid monolayer of lipid bodies. The density of L3 in the surface layer of individual lipid bodies does not change during seedling growth; therefore, the decrease in the amount of L3 can be attributed to a decrease in the number of lipid bodies rather than to selective removal of protein components from the surface of all lipid bodies. Thus, L3 is apparently degraded at the same time as the matrix lipid of each lipid body. Unlike lipase, L3 does not appear to be transferred to other cellular compartments such as vacuoles during late stages of seedling growth.     

Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.

We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems.     

Conserved core of amyloid fibrils of wild type and A30P mutant α-synuclein

The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein α-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected high-resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of β-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates.     

Unfolding and conformational distributions during protein precipitation

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.