Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction

TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-kappaB (NF-kappaB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-kappaB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-kappaB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50 approximately 30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-kappaB activation and subsequent cellular events.     

伤害信号分子及其信号转导

伤害对于植物是一种常见的环境刺激。目前,对伤害刺激产生的防御反应及其机理都有了较为广泛的研究。简述了目前已确定的参与伤害反应的信号分子：寡糖素,系统素,脱落酸,茉莉酸,乙烯和电信号等,并初步探讨了伤害信号分子的信号转导途径。     

Calcium signals in growth factor signal transduction

There is a substantial amount of information which has been obtained concerning the effects of growth factors on [Ca²⁺]_i in proliferating cells. A number of different mitogens are known to induce elevations in [Ca²⁺]_i and some characterization of the Ca²⁺ response to different classes of mitogens has been obtained. In addition, much is known about whether the Ca²⁺ response to a particular growth factor occurs as the result of an influx of external Ca²⁺ or a mobilization of internal Ca²⁺ stores. In addition, a considerable amount of information is available on the mechanism by which the Ins(1,4,5)P₃-sensitive internal Ca²⁺ store takes up and releases Ca²⁺. However, there is still a large deficiency in our information concerning other Ca²⁺ stores in proliferating cells as well as in our knowledge of the mechanisms for regulating Ca²⁺ entry pathways. Much more data addressing these issues exists for other types of agonist-stimulated cells, and we have discussed much of it in this review article. While the wealth of data in nonproliferating cells provides some indications of what mechanisms might be involved in the growth factor-induced changes in [Ca²⁺]_i, it is clear that much work must be done in proliferating cells to fully understand how external factors such as growth factors control [Ca²⁺]_i. In addition, much work remains to be done in identifying the mechanisms for the internal control of [Ca²⁺]_i as cells move through the cell cycle and in identifying the role that these changes in [Ca²⁺]_i may play throughout the cell cycle.     

Inhibitory effects of biseokeaniamide A against lipopolysaccharide-induced signal transduction

Lipopolysaccharides (LPS) are associated with various inflammatory diseases; therefore, the inhibition of LPS-induced nitric oxide (NO) production may have extensive therapeutic applications. We searched for inhibitors of NO production in the LPS-stimulated murine macrophage-like cell line RAW264.7 from MeOH extracts of marine organisms. The MeOH extract of the marine cyanobacterium Okeania sp., collected in Okinawa, Japan, showed inhibitory activity. Biseokeaniamide A was isolated from the MeOH extract by chromatographic separation. Biseokeaniamide A inhibited NO production without cytotoxicity. It reduced inducible nitric oxide synthase levels and suppressed the expression of IL-1β in LPS-stimulated RAW264.7 cells. Biseokeaniamide A did not inhibit IκBα degradation but inhibited IκBα expression. Thus, biseokeaniamide A, a naturally occurring lipopeptide, was identified as a selective inhibitor of LPS signal transduction.     

Zinc as a possible mediator of signal transduction in T lymphocytes

Our recent findings indicate that phorbol esters, the specific activators of protein kinase C induce the translocation of heavy metals (mostly: zinc) from the nucleus and mitochondria to the cytosol and microsomes of T lymphocytes. Phorbol ester treatment impairs the action of Ca-ionophores, this effect is mediated by intracellular heavy metal ions (most probably: by zinc). Zinc activates cytosolic protein kinase C, increases its affinity towards phorbol esters and contributes to its binding to plasma membranes. These results suggest that zinc may play a role in the "cross-talk" of second messengers and hence in signal transduction in T lymphocytes.     

Inter-helical hydrogen bonds are essential elements for intra-protein signal transduction: the role of Asp115 in bacteriorhodopsin transport function

The behavior of the D115A mutant was analyzed by time-resolved UV-Vis and Fourier transformed infrared (FTIR) spectroscopies, aiming to clarify the role of Asp115 in the intra-protein signal transductions occurring during the bacteriorhodopsin photocycle. UV-Vis data on the D115A mutant show severely desynchronized photocycle kinetics. FTIR data show a poor transmission of the retinal isomerization to the chromoprotein, evidenced by strongly attenuated helical changes (amide I), the remarkable absence of environment alterations and protonation/deprotonation events related to Asp96 and direct Schiff base (SB) protonation form the bulk. This argues for the interactions of Asp115 with Leu87 (via water molecule) and Thr90 as key elements for the effective and vectorial proton path between Asp96 and the SB, in the cytoplasmic half of bacteriorhodopsin. The results strongly suggest the presence of a regulation motif enclosed in helices C and D (Thr90-Pro91/Asp115) which drives properly the dynamics of helix C through a set of interactions. It also supports the idea that intra-helical hydrogen bonding clusters in the buried regions of transmembrane proteins can be potential elements in intra-protein signal transduction.     

Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal transduction

We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two VPAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys(195)). VIP had a lower affinity for R188Q, R188L, K195Q, and K195I VPAC(1) receptors than for VPAC(1) receptors. [Asn(3)] VIP and [Gln(3)] VIP had lower affinities than VIP for VPAC(1) receptors but higher affinities for the mutant receptors; the two basic amino acids facilitated the introduction of the negatively charged aspartate inside the transmembrane domain. The resulting interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [Gln(3)] VIP were partial agonists at VPAC(1) receptors; 2/VIP did not fully activate the K195Q, K195I, R188Q, and R188L VPAC(1) receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn(3)] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q and R188L VPAC(1) receptors; 3/the [Asp(3)] negative charge did not contribute to the recognition of the VIP(1) antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27)] VIP ()/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC(1) receptor, the Asp(3) side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2.     

S-adenosylmethionine may not be essential for signal transduction during bacterial chemotaxis.

We previously showed that a mutant strain of Salmonella typhimurium completely deficient in both the chemoreceptor methylating (CheR) and demethylating (CheB) enzymes can still exhibit chemotaxis to aspartate and other attractants (J. Stock, A. Borczuk, F. Chiou, and J. E. B. Burchenal, Proc. Natl. Acad. Sci. USA 82:8364-8368, 1985). We used this cheR cheB mutant to examine the possibility of an additional requirement for S-adenosylmethionine in chemotaxis besides its role in chemoreceptor methylation. A metE mutation was transduced into a cheR cheB double mutant, and the cells were starved for methionine. Despite the fact that intracellular S-adenosylmethionine dropped from approximately 100 microM to less than 0.2 microM, chemotaxis was largely unaffected. In contrast, a corresponding cheR+ cheB+ metE mutant completely lost its chemotaxis ability after being starved for methionine. We conclude from this observation that the primary requirement for S-adenosylmethionine during bacterial chemotaxis is in the methylation of receptor proteins.     

Aminopeptidase N/CD13 is directly linked to signal transduction pathways in monocytes

In the present study, we characterized in monocytes the rise in [Ca(2+)](i) evoked by monoclonal antibodies (mAbs) to aminopeptidase N (APN)/CD13, showing a two-phase calcium increase with a small-belled [Ca(2+)](i) rise due to the release of calcium from intracellular stores and a more sustained plateau due to the influx of calcium from the extracellular environment. Tyrosine kinase inhibitors were able to inhibit the rise in [Ca(2+)](i) induced by ligation APN/CD13, as were inhibitors of the phosphatidylinositol 3-kinase. For the first time we can show that mAbs to APN/CD13 provoke phosphorylation of the mitogen-activated protein kinases ERK1/2, JNK, and p38. Furthermore, we show that mRNA of the chemotactic cytokine IL-8 is upregulated under the influence of APN/CD13 ligation. Although the in vivo ligand as well as possible cooperating membrane molecules remains to be identified, our results suggest that the membrane ectoenzyme APN/CD13 is a novel signal transduction molecule in monocytes.     

Lipid rafts are required for efficient signal transduction by CD1d

Plasma membranes of eukaryotic cells are not uniform, possessing distinct cholesterol- and sphingolipid-rich lipid raft microdomains which constitute critical sites for signal transduction through various immune cell receptors and their co-receptors. CD1d is a conserved family of major histocompatibility class I-like molecules, which has been established as an important factor in lipid antigen presentation to natural killer T (NKT) cells. Unlike conventional T cells, recognition of CD1d by the T cell receptor (TCR) of NKT cells does not require CD4 or CD8 co-receptors, which are critical for efficient TCR signaling. We found that murine CD1d (mCD1d) was constitutively present in the plasma membrane lipid rafts on antigen presenting cells, and that this restricted localization was critically important for efficient signal transduction to the target NKT cells, at low ligand densities, even without the involvement of co-receptors. Further our results indicate that there may be additional regulatory molecule(s), co-located in the lipid raft with mCD1d for NKT cell signaling.     

Src family kinases are required for integrin but not PDGFR signal transduction

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors.