Farnesyltransferase inhibitor improved survival following endotoxin challenge in mice

Endotoxemia plays an important role in the pathogenesis of sepsis and is accompanied by dysregulated apoptosis of immune and non-immune cells. Treatment with statins reduces mortality in rodent models of sepsis and endotoxemia. Inhibition of protein isoprenylation, including farnesylation, has been proposed as a mechanism to mediate the lipid-lowering-independent effects of statins. Nonetheless, the effects of the inhibition of isoprenylation have not yet been studied. To investigate the role of farnesylation, we evaluated the effects of farnesyltransferase inhibitor and statin on survival following lipopolysaccharide (LPS) challenge in mice. Both simvastatin (2 mg/kg BW) and FTI-277 (20 mg/kg BW) treatment improved survival by twofold after LPS injection, as compared with vehicle alone (p < 0.01). LPS-induced cleavage (activation) of caspase-3, an indicator of apoptotic change, and increased protein expression of proapoptotic molecules, Bax and Bim, and activation of c-Jun NH₂-terminal kinase (JNK/SAPK) in the liver and spleen were attenuated by both simvastatin and FTI-277. These results demonstrate that farnesyltransferase inhibitor as well as statin significantly reduced LPS-induced mortality in mice. Our findings also suggest that inhibition of protein farnesylation may contribute to the lipid-lowering-independent protective effects of statins in endotoxemia, and that protein farnesylation may play a role in LPS-induced stress response, including JNK/SAPK activation, and apoptotic change. Our data argue that farnesyltransferase may be a potential molecular target for treating patients with endotoxemia.     

The acute phase response stimulates the expression of angiopoietin like protein 4

The acute phase response is characterized by elevations in serum triglyceride levels due to both an increase in hepatic VLDL production and a delay in the clearance of triglyceride rich lipoproteins secondary to a decrease in lipoprotein lipase (LPL) activity. Recently there has been a marked increase in our understanding of factors that regulate LPL activity. GPIHBP1 facilitates the interaction of LPL and lipoproteins thereby allowing lipolysis to occur. Angiopoietin like proteins (ANGPTL) 3 and 4 inhibit LPL activity. In the present study, treatment of mice with LPS, an activator of TLR4 and a model of Gram-negative infections, did not alter the expression of GPIHBP1 in heart or adipose tissue. However, LPS decreased the expression of ANGPTL3 in liver and increased the expression of ANGPTL4 in heart, muscle, and adipose tissue. Serum ANGPTL4 protein levels were markedly increased at 8 and 16 h following LPS treatment. Administration of zymosan, an activator of TLR2 and a model of fungal infections, also increased serum ANGPTL4 protein and mRNA levels in liver, heart, muscle, and adipose tissue. Finally, treatment of 3T3-L1 adipocytes with LPS or cytokines (TNF alpha, IL-1 beta, and interferon gamma) stimulated ANGPTL4 expression. These studies demonstrate that ANGPTL4 is a positive acute phase protein and the increase in ANGPTL4 could contribute to the hypertriglyceridemia that characteristically occurs during the acute phase response by inhibiting LPL activity.     

Structural Basis for the Biological Effects of Pr(III) Ions: Alteration of Cell Membrane Permeability

The biological effects of rare-earth ions on the organism have been studied using Pr³⁺ as a probe ion and Escherichia coli cell as a target. Atomic force microscopy (AFM) observation of the surface of E. coli cells shows that the presence of Pr³⁺ substantially changes the structure of the outer membrane. By induced coupled plasma-mass spectrometry (ICP-MS), more Cu²⁺ was found in the cells grown in the presence of Pr³⁺, indicating changes of cell permeability. Using energy dispersive X-ray spectroscopy (EDX), Ca²⁺ is found on the outer surface of the original cell. It is proposed that Pr³⁺ can replace Ca²⁺ from the binding sites because of their close ionic radii and similar ligand speciality.     

Induction of TNF-α by LPS in Schwann Cell is Regulated by MAPK Activation Signals

Mitogen-activated protein kinases (MAPKs) are important mediators of cytokine expression and are critically involved in the immune response. The lipopolysaccharide (LPS) of gram-negative bacteria induces the expression of cytokines and proinflammatory genes via the toll-like receptor 4 (TLR4) signaling pathway in diverse cell types. In vivo, Schwann cells (SCs) at the site of injury may also produce tumor necrosis factor-- α (TNF-α). However, the precise mechanisms of TNF-α synthesis are still not clear. The purpose of the present study was to elucidate the underlying molecular mechanisms in the cultured SCs for its ability to activate the MAPKs and TNF-α gene, in response to LPS. Using enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS stimulated the synthesis of TNF-α in a concentration- and time-dependent manner. Intracellular location of TNF-α was detected under confocal microscope. Moreover, LPS activated extracellular signal-regulated kinase (ERK1/2), P38 and stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and induced their phosphorylation. LPS-elicited SCs TNF-α production was also drastically suppressed by PD98059 (ERK inhibitor), SB202190 (P38 inhibitor), or SP600125 (SAPK/JNK inhibitor). Additionally, the expression of CD14 and TLR4 was examined by RT–PCR. It was demonstrated that the expression of CD14, TLR4 was crucial for the SCs responses to LPS. In conclusion, the results provide novel mechanisms for the response of SCs to LPS stimulation, through MAPKs signaling pathways. Chun Cheng and Yongwei Qin contributed equally to this work.     

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

蝮蛇毒蛋白C激活物对内毒素性离体大鼠心脏功能的影响

目的研究蝮蛇毒蛋白C激活物(PCA)组分对大鼠内毒素(LPS)性心肌损伤作用的影响。方法取SD雄性大鼠32只随机分成正常对照组、LPS组、PCA组和PCA LPS组,用Krebs-Henseleit(K-H)液对大鼠离体心脏行主动脉逆灌。在相应时点记录HR、LVSP、LVEDP、LVDP、 dp/dtmax、-dp/dtmax的变化,并测定冠脉流出液中的超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量。结果PCA明显减轻LPS诱导的心功能改变并抑制心肌SOD活性降低和MDA的升高(P<0.01)。结论PCA对内毒素诱导的心肌损伤改善作用明显,其机制可能是通过保护血管内皮功能,改善微循环,稳定心肌酶活性和膜相结构等途径有关。     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Generation and characterization of a lipopolysaccharide-specific murine monoclonal antibody to <Emphasis Type="Italic">Proteus vulgaris</Emphasis> OX19

The three strains of non-pathogenic Proteus species namely, Proteus vulgaris OX2, P. vulgaris OX19 and Proteus mirabilis OXK used in the Weil–Felix test are the group-specific cross-reactive antigens for Rickettsia and Orientia species. Earlier studies have revealed that the group specific and cross-reactive antigens responsible for the Weil–Felix test lie mostly in the lipopolysaccharide (LPS) moiety of the bacterial cell wall [Amano et al. (1993a) Infect Immun 61:4350–4355, (1993b) Microbiol Immunol 37:927–933, (1998) Infect Immun 66:923–926]. The three Proteus strains (OX2, OX19 and OXK) were used to raise murine monoclonal antibodies (MAbs) by hybridoma technology. Several MAb-producing hybridomas could be stabilized following limiting dilution. Affinity and specificity of these MAbs were checked by indirect ELISA using a battery of homologous and heterologous antigens including LPS. Amongst these, one MAb was found to be specific for P. vulgaris OX19 LPS. Since the Weil–Felix reaction is based on the cross-reactivity between the LPS based epitopes, this MAb could be of potential use in mapping of epitopes on the cross-reactive LPS and may also be useful as a potential diagnostic reagent.