The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.     

Transient Receptor Potential Ankyrin 1 Receptor Activation In Vitro and In Vivo by Pro-tussive Agents: GRC 17536 as a Promising Anti-Tussive Therapeutic

Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate in vitro direct functional activation of TRPA1 receptor by citric acid which is routinely used to evoke cough in preclinical and clinical studies. We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca⁺² influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1.     

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using ¹³C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO₂ via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Immunoregulation through IL-10 gene expression and the fate of cytotoxic T lymphocyte-mediated tumor immunotherapy

Gene analysis of tumor associated antigens revealed that tumor antigens are all normal gene product. Inducing tumor reactive cytotoxic T lymphocytes (CT) in the patients is same as inducing autoimmunity in the patients. Immunosuppressive cytokine interleukin-10 (IL-10) plays an important role in maintaining homeostasis or tolerance. To break the tumor tolerance, blocking and IL-10 secretion or intervention in the pathways of IL-10 gene activation is indeed important.     

Chemotaxis of chemolithotrophic Thiobacillus ferrooxidans toward thiosulfate

Abstract The chemotactic response of Thiobacillus ferrooxidans toward thiosulfate was observed. The traditional assay technique was modified by direct microscopic enumeration of cells which moved into the attractant solution. The optimum concentration shown by thiosulfate-grown cells, tetrathionate-grown cells as well as iron-grown cells was 10³ times the optimum concentration shown by cells grown on elemental sulfur. Iron-grown cells which lack thiosulfate-oxidizing activity showed increased accumulation at optimum concentration as compared to cells grown on elemental sulfur and other reduced sulfur compounds. This indicated the constitutive nature of chemotaxis by T. ferrooxidans toward thiosulfate.     

Characterization of guanylyl cyclase in purified myelin

This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latters being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed K_m values of 0.258 mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. V_max values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath. Special issue dedicated to Dr. Marion E. Smith.     

7-Dehydrositosterol from Rauwolfia serpentina

7-Dehydrositosterol has been isolated from the roots of Rauwolfia serpentina.     

Effect of iron on siderophore production and on outer membrane proteins ofRhizobium leguminosarum IARI 102

Rhizobium leguminosarum IARI 102 produced a phenolate type siderophore (a derivative of 2,3-DHBA) under iron-limited conditions. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but repressed the production of the iron-chelating compound. Iron level of culture medium also had a significant role in the composition of outer membrane proteins ofR. leguminosarum IARI 102. Maximum iron uptake was observed only in the presence of its own siderophore.     

MSH receptors in immortalized human epidermal keratinocytes: A potential mechanism for coordinate regulation of the epidermal-melanin unit

Receptors for melanotropin (MSH) were found to be expressed by immortalized primary human epidermal keratinocytes (RHEK-1). Using ¹²⁵I-βMSH as a probe, the MSH receptors from mouse melanoma cells and human keratinocytes were found to be remarkably similar. In each cell line, there were high and low affinity receptors, with the high affinity classes showing positive cooperativity. Competition of ¹²⁵l-βMSH for binding with non-radioactive MSH revealed similar profiles. Cross-linking studies, followed by gel electrophoresis and autoradiography, showed almost identical gel migration patterns. Both cell types expressed internal as well as plasma membrane binding sites. MSH receptors on both cell types were up-regulated by ultraviolet light and by MSH itself. Although the function of MSH receptors expressed by the immortalized keratinocytes is unknown, the results are consistent with recent reports that proliferation of epidermal keratinocytes is stimulated by MSH and that proopiomelanocortin genes are expresed in the epidermis. These results support a model in which keratinocytes and melanocytes, interacting in an “epidermal-melanin unit,” each respond to UV light signals with increased MSH receptor activity. © 1993 Wiley-Liss, Inc.