Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A₂ (sPLA₂), group IIA (sPLA₂-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA₂ enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA₂s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA₂-V, and to a lesser extent that of sPLA₂-IID, -IIE, and -IIF, were increased, whereas that of sPLA₂-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA₂-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA₂-IIA inhibitor that turned out to inhibit sPLA₂-IID, -IIE, -V and -X as well. In contrast, sPLA₂-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA₂s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

A proteomic investigation into the human cervical cancer cell line HeLa treated with dicitratoytterbium (III) complex

Lanthanides have been reported to induce apoptosis in cancer cell lines. Human cervical cancer cell line HeLa was found to be more sensitive to dicitratolanthanum (III) complex ([LaCit₂]³⁻) than other cancer cell lines. However, the effect and mechanism of dicitratoytterbium (III) complex ([YbCit₂]³⁻) on HeLa cells is unknown. Using biochemical and comparative proteomic analyses, [YbCit₂]³⁻ was found to inhibit HeLa cell growth and induce apoptosis. Similar to the effects of [LaCit₂]³⁻, proteomics results from [YbCit₂]³⁻-treated cells revealed profound changes in proteins relating to mitochondria and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [YbCit₂]³⁻-induced apoptosis. This was confirmed by the decreased mitochondrial transmembrane potential and the increased generation of reactive oxygen species in [YbCit₂]³⁻-treated cells. Western blot analysis showed that [YbCit₂]³⁻-induced apoptosis was accompanied by the activation of caspase-9 and specific proteolytic cleavage of PARP, leading to an increase in the pro-apoptotic protein Bax and a decrease in the anti-apoptotic protein Bcl-2. These results suggest a mitochondrial pathway of cell apoptosis in [YbCit₂]³⁻-treated cells, which will help us understand the molecular mechanisms of lanthanide-induced apoptosis in tumor cells.     

Reduction of gene expression by a hairpin-loop structured oligodeoxynucleotide: Alternative to siRNA and antisense

Background

We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection.

Methods and results

Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5′ untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out.

General significance

Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Nectin-3 expression is elevated in limbal epithelial side population cells with strongly expressed stem cell markers

Corneal epithelial stem cells (CESCs) are essential for maintaining the ocular surface. However, the lack of surface markers for CESCs remains a serious obstacle in the identification of CESCs. Previously, we showed that rabbit limbal epithelial side population (rLE-SP) cells exhibited stem cell phenotypes including increased expression of CD61, a marker for mouse hematopoietic stem cells. Here, we demonstrate that nectin-3, an immunoglobulin-like cell-cell adhesion molecule, is highly expressed in rLE-SP cells. Additionally, nectin-3⁺ cells were significantly enriched among CD61⁺rLE-SP cells as compared to CD61⁻rLE-SP cells. In mouse bone marrow side population cells, a correlation between expression of nectin-3 and CD61 was also observed. These data strongly suggest that nectin-3 may contribute to the identification of CESCs.     

Kinetic and X-ray structural evidence for negative cooperativity in substrate binding to nicotinate mononucleotide adenylyltransferase (NMAT) from Bacillus anthracis

Biosynthesis of NAD(P) in bacteria occurs either de novo or through one of the salvage pathways that converge at the point where the reaction of nicotinate mononucleotide (NaMN) with ATP is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate. This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT), which is essential for bacterial growth, making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed to exhibit negative cooperativity, i.e. Hill coefficients < 1.0. Negative cooperativity in binding is supported by the results of X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN- and NaAD-bound complexes were determined to resolutions of 2.50 Å, 2.60 Å and 1.75 Å, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer, supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model in which the binding affinity of substrates to the first monomer of NMAT is stronger than that to the second, and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has not been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD(P) biosynthetic pathway may occur, in part, at the reaction catalyzed by NMAT.