Endothelial cell junctions and the regulation of vascular permeability and leukocyte transmigration.

The endothelial lining of the vasculature forms the physical barrier between the blood and underlying tissues. Junctions between adjacent endothelial cells are dynamically modulated to sustain vascular homeostasis and to support the transendothelial migration of leukocytes during inflammation. A variety of factors initiate intracellular signaling pathways that regulate the opening and resealing of junctional complexes. This review focuses on three primary signaling pathways initiated within endothelial cells by the binding of vasoactive factors and leukocyte adhesion: Rho GTPases, reactive oxygen species, and tyrosine phosphorylation of junctional proteins. These pathways converge to regulate junctional permeability, either by affecting the stability of junctional proteins or by modulating their interactions. Although much progress has been made in understanding the relationships of these pathways, many questions remain to be answered. A full understanding of the signaling cascades that affect endothelial junctions should identify novel therapeutic targets for diseases that involve excessive permeability or inappropriate leukocyte infiltration into tissues.

Signal transduction triggered by lipid A-like molecules in 70Z/3 pre-B lymphocyte tumor cells.

The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-kappaB, and transcription of kappa light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-kappaB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1-50 microM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-kappaB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-kappaB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-kappaB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-kappaB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-kappaB.

Accumulation of a lipid A precursor lacking the 4'-phosphate following inactivation of the Escherichia coli lpxK gene.

The lpxK gene has been proposed to encode the lipid A 4'-kinase in Escherichia coli (Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997) J. Biol. Chem. 272, 21855-21864). In cell extracts, the kinase phosphorylates the 4'-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters. msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an Omega-chloramphenicol cassette into msbA also halts transcription of lpxK. We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy of lpxK. This mutation is complemented at 30 degreesC by a hybrid plasmid with a temperature-sensitive origin of replication carrying lpxK+. When this strain (designated TG1/pTAG1) is grown at 44 degreesC, the plasmid bearing the lpxK+ is lost, and the phenotype of an lpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44 degreesC, consistent with lpxK being an essential gene. Furthermore, 4'-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4'-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44 degreesC. The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by 1H NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis in E. coli. Homologs of lpxK are present in the genomes of other Gram-negative bacteria.

Identification of the gene encoding the Escherichia coli lipid A 4'-kinase. Facile phosphorylation of endotoxin analogs with recombinant LpxK.

The genes for seven of nine enzymes needed for the biosynthesis of Kdo2-lipid A (Re endotoxin) in Escherichia coli have been reported. We have now identified a novel gene encoding the lipid A 4'-kinase (the sixth step of the pathway). The 4'-kinase transfers the gamma-phosphate of ATP to the 4'-position of a tetraacyldisaccharide 1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1,4'-bis-phosphate (lipid IVA). The 4'-phosphate is required for the action of distal enzymes, such as Kdo transferase and also renders lipid A substructures active as endotoxin antagonists or mimetics. Lysates of E. coli generated using individual lambda clones from the ordered Kohara library were assayed for overproduction of 4'-kinase. Only one clone, [218]E1D1, which directed 2-2.5-fold overproduction, was identified. This construct contains 20 kilobase pairs of E. coli DNA from the vicinity of minute 21. Two genes related to the lipid A system map in this region: msbA, encoding a putative translocator, and kdsB, the structural gene for CMP-Kdo synthase. msbA forms an operon with a downstream, essential open reading frame of unknown function, designated orfE. orfE was cloned into a T7 expression system. Washed membranes from cells overexpressing orfE display approximately 2000-fold higher specific activity of 4'-kinase than membranes from cells with vector alone. Membranes containing recombinant, overexpressed 4'-kinase (but not membranes with wild-type kinase levels) efficiently phosphorylate three DS-1-P analogs: 3-aza-DS-1-P, base-treated DS-1-P, and base-treated 3-aza-DS-1-P. A synthetic hexaacylated DS-1-P analog, compound 505, can also be phosphorylated by membranes from the overproducer, yielding [4'-32P] lipid A (endotoxin). The overexpressed lipid A 4'-kinase is very useful for making new 4'-phosphorylated lipid A analogs with potential utility as endotoxin mimetics or antagonists. We suggest that orfE is the structural gene for the 4'-kinase and that it be redesignated lpxK.

Novel requirements in peripheral structures of the extended satellite 2 hammerhead.

A number of RNAs that infect plants self-cleave using a domain called the hammerhead. The consensus plant hammerhead has three base paired stem structures. Stems I and III flank the cleaved phosphodiester and are connected to stem II by two unpaired and highly conserved sequences. We demonstrated previously that satellite 2 transcripts from the newt, Notophthalmus viridescens, use a modified hammerhead structure for self-cleavage. Here we show that hammerheads with similar modifications occur in satellite 2 from species representing two additional families of salamanders (Ambystoma talpoideum and Amphiuma tridactylum). The distinctive features of the modified satellite 2 hammerheads include unusually short stem III regions and internally looped extensions to stem I that are required for cleavage. However, despite the strict nucleotide requirement in the internal stem I loop of the newt hammerhead, the details of the stem I extensions differ in the three salamanders. An analysis of chimeric transcripts indicated that, in order for a specific stem I extension to be functional, it must be compatible with other regions of the modified satellite 2 hammerhead. One such region is stem II, and a mutational analysis has confirmed that there are specific sequence and/or structural requirements in stem II.