Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E(2) in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA(1)) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA(1) also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA(1) and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA(1) and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA(1)-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA(1) decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA(1) offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.     

Changes of nitric oxide level at different stages of primary open-angle glaucoma

The contents of NO stable metabolites in tears, and blood serum of the patients with different stages of open-angle glaucoma have been studied. The increasing of NO stable metabolite contents was found in tears, aqueous humor and blood serum during glaucoma progression. The depletion of glaucoma pathogenesis oxidative link was found at the terminal stage of the disease. The results of our investigation are proposed to be used for glaucoma diagnostic.     

Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: functional differences between sphingosine kinase 1a and 1b

Sphingosine kinase 1 catalyses the formation of the bioactive lipid, sphingosine 1-phosphate and is a target for anti-cancer agents. We demonstrate here that 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi, also referred to as SKI-II), FTY720 (Fingolimod), and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 activity with distinct kinetics, indicating that these compounds exhibit different binding modalities with sphingosine kinase 1. Thus, SKi is a mixed inhibitor of sphingosine and ATP binding, whereas FTY720 is competitive with sphingosine and uncompetitive with ATP, and (S)-FTY720 vinylphosphonate is uncompetitive with sphingosine and is a mixed inhibitor with respect to ATP. A novel 'see-saw' model is proposed for the binding of inhibitor to catalytic and allosteric sites, the latter dependent on substrate binding, that provides an explanation for the different inhibitor kinetics. In addition, we demonstrate that the expression level and properties unique to an N-terminal 86 amino-acid isoform variant of sphingosine kinase 1 (SK1b) in prostate cancer cells reduce its sensitivity to SKi-induced proteasomal degradation in comparison to SK1a, i.e. these two N-terminal variants of sphingosine kinase 1 (SK1a and SK1b) have different properties. The reduced sensitivity of SK1b to proteasomal degradation in response to SKi is translated into specific changes in ceramide and S1P levels that leads to apoptosis of androgen-sensitive but not androgen-independent LNCaP prostate cancer cells. Therefore, our proposed 'see-saw' model might be usefully employed in the design of sphingosine kinase inhibitors to promote apoptosis of chemotherapeutic resistant cancer cells.     

A non-destructive sensitive reagent for lipid detection on TLC plates

The aqueous solutions of 3 acidic and 14 basic dyes used as reagents for detection of lipids without their destruction are tested on plates for highly effective TLCh with the silicagel layer fixed by the silicic acid sol. It is shown that 0.002% solution of basic fuchsin in 1% boric acid reveals neutral, phospho- and glycolipids with the sensitivity compared with such in detection of sulphuric acid. It is established that detection of lipids by this reagent with the use of TLCh does not change the composition of their fatty acids.