Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release

Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A2, the initial rate-limiting step. In this study, ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1beta (IL-1beta), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, ceramide kinase, was activated in response to IL-1beta and A23187. RNA interference targeted to ceramide kinase specifically down-regulated ceramide kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1beta and A23187. Down-regulation of ceramide kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that ceramide kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A2 activation. This study identifies previously unknown roles for ceramide kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.     

Ultrastructure of male sex pheromone glands in abdominal tergites of five Lutzomyia sandfly species (Diptera: Psychodidae)

The fine structure of male sex pheromone disseminating structures on abdominal segments of five species of Lutzomyia sandflies (Diptera: Psychodidae) was analyzed. Scanning electron microscopy showed that in Lutzomyia cruzi [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia longipalpis [Mem. Inst. Oswaldo Cruz 4 (1912) 84], the disseminating structures are located in pale spots on abdominal tergites IV or III and IV and are morphologically similar, appearing as small round cuticular elevations with a central pore. Observation of abdominal tergites of L. longipalpis pupae showed that the spots, but not the structures, are already present in this stage. In Lutzomyia lenti [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia carmelinoi [Mem. Inst. Oswaldo Cruz 81 (1986) 323] adult males, the disseminating structures are present on tergal segments V and VI, where pale spots could not be observed, and appear as apple-like elevations with a central pore. In Lutzomyia renei, a single disseminating structure is found at the anterior region of tergal segment VI. Transmission electron microscopy was used to analyze the gland cell fine structure in L. cruzi. Several class III gland cells are located side by side in the fourth abdominal segment. Each epidermal secretory cell contains a small reservoir and a short outlet channel through the cuticle.     

Cellular junctions of myelinated nerves (Review)

Myelinated nerves are specifically designed to allow the efficient and rapid propagation of action potentials. Myelinating glial cells contain several types of cellular junctions that are found between the myelin lamellas themselves in specialized regions of non-compact myelin and between the myelin membrane and the underlying axon. These include most of the junctional specializations found in epithelial cells, including tight, gap and adherens junctions. However, whereas in epithelial cells these junctions are formed between different cells, in myelinating glia these so called autotypic junctions are found between membrane lamellae of the same cell. In addition, myelinating glial cells form a heterotypic septate-like junction with the axon around the nodes of Ranvier and, in the peripheral nerve system, contact the basal lamina, which surrounds myelinating Schwann cells. This short review discusses the structure, molecular composition and function of the junctions present in myelinating cells, concentrating on the axo-glial junction.     

Lipid mediators of angiogenesis and the signalling pathways they initiate

Investigations carried out over the past 3 years have implicated a key role for sphingosine 1-phosphate (SPP) in angiogenesis and blood vessel maturation. SPP is capable of inducing almost every aspect of angiogenesis and vessel maturation in vitro, including endothelial cell chemotaxis, survival, proliferation, capillary morphogenesis and adherence antigen deployment, as well as stabilizing developing endothelial cell monolayers and recruitment of smooth muscle cells to maturing vessels. Acting in conjunction with protein angiogenic factors, SPP induces prolific vascular development in many established models of angiogenesis in vivo. Thus, SPP is a unique, potent and multifaceted angiogenic agent. While SPP induces angiogenic effects by ligating members of the endothelial differentiation gene (EDG) G-protein-coupled family of receptors, recent studies suggest that endogenously produced SPP may also account for the ability of tyrosine kinase receptors to induce cell migration. Thus, SPP provides a clear link between tyrosine kinase and G-protein-coupled receptor agonists involved in the angiogenic response. However, the mechanisms by which SPP exerts its effects on vascular cells remain unclear, conflicting and controversial. Precise definition of the signalling pathways by which SPP induces specific aspects of the angiogenic response promises to lead to new and effective therapeutic approaches to regulate angiogenesis at sites of tissue damage, neoplastic transformation and inflammation. This review will trace the discovery of SPP as a novel angiogenic factor as it outlines present information on the signalling pathways by which SPP induces its effects on cells of the developing vascular bed.     

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.     

Sphingosine-1-phosphate: an enigmatic signalling lipid

The evolutionarily conserved actions of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), in yeast, plants and mammals have shown that it has important functions. In higher eukaryotes, S1P is the ligand for a family of five G-protein-coupled receptors. These S1P receptors are differentially expressed, coupled to various G proteins, and regulate angiogenesis, vascular maturation, cardiac development and immunity, and are important for directed cell movement.     

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.     

Why do cytotoxic T lymphocytes fail to eliminate hepatitis C virus? Lessons from studies using major histocompatibility complex class I peptide tetramers.

Hepatitis C virus (HCV) infection is a major public health problem, affecting an estimated 3% of the world's population, and over 10% in some countries. Infection in most cases becomes persistent, and can lead to hepatic inflammation, fibrosis and liver failure. The T lymphocyte reponse, in particular that mediated by cytotoxic T lymphocytes (CTLs), is likely to be involved in determining the outcome of infection, although its overall role is not clear. The use of major histocompatibility complex (MHC) class I peptide tetrameric complexes (tetramers) to study antiviral CTL responses has revolutionized our approach to the study of human infection. We have used a panel of MHC class I tetramers to analyse immune responses in HCV-infected individuals at various stages of disease. We find that the CTL response against HCV is vigorous in its early phases but dwindles over time both in terms of lymphocyte number and function. A number of potential explanations for this 'CTL failure' are discussed.     

Spectrometric studies on stability of tenuazonic acid (TeA) solution in organic solvents

The stability of tenuazonic acid solution at different temperatures and storage times was studied using methanol, methanol-water (8:2 v/v), benzene and benzene-acetonitrile (98:2 v/v) as solvents. Solutions were analysed by a spectrometric method TeA U.V.-spectrum was recorded. Results indicated that the optimum temperature for long-time storage period of tenuazonic acid solution in any solvent assayed is -20°C. Benzene and benzene-acetonitrile (98:2 v/v) could be advised to make tenuazonic acid solution which will be stored less than 2 months at 4°C. Methanol and methanolwater (8:2 v/v) are not recommended because a low stability of TeA solution in this solvents.