Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V+) and uninfected (V-) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V+ compared with V- isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V+ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V+ and V- isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Differential dissolved protein expression throughout the life cycle of Giardia lamblia.

Giardia lamblia (G. lamblia) has a simple life cycle that alternates between a cyst and a trophozoite, and this parasite is an important human and animal pathogen. To increase our understanding of the molecular basis of the G. lamblia encystment, we have analyzed the soluble proteins expressed by trophozoites and cysts extracted from feces by quantitative proteomic analysis. A total of 63 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and were categorized as cytoskeletal proteins, a cell-cycle-specific kinase, metabolic enzymes and stress resistance proteins. Importantly, we demonstrated that the expression of seven proteins differed significantly between trophozoites and cysts. In cysts, the expression of three proteins (one variable surface protein (VSP), ornithine carbamoyltransferase (OTC), ß-tubulin) increased, whereas the expression of four proteins (14-3-3 protein, α-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein disulfide isomerase 2 (PDI-2)) decreased significantly when compared with the levels of these proteins in trophozoites. The mRNA expression patterns of four of these proteins (OTC, α-tubulin, GAPDH, VSP) were similar to the expression levels of the proteins. These seven proteins appear to play an important role in the completion of the life cycle of G. lamblia.

The effect of magnolol on the Toll-like receptor 4/nuclear factor κB signaling pathway in lipopolysaccharide-induced acute lung injury in mice.

Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis has been reported to have anti-inflammatory properties. The purpose of this study was to evaluate the effect of magnolol on acute lung injury induced by lipopolysaccharide in mice. Male BALB/c mice were pretreated with dexamethasone or magnolol 1 h before intranasal instillation of lipopolysaccharide (LPS). 7 h after LPS administration, the myeloperoxidase in lung tissues, lung wet/dry weight ratio and inflammatory cells in the bronchoalveolar lavage fluid were determined. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in the bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay (ELISA). The extent of phosphorylation of nuclear factor of inhibitory kappa B alpha (IκB-α), nuclear factor kappa-B (NF-κB) p65 and the expression of Toll-like receptor-4 (TLR4) were detected by western blot. The results showed that magnolol markedly attenuated the histological alterations in the lung; reduced the number of total cells, neutrophils, and macrophages in the bronchoalveolar lavage fluid; decreased the wet/dry weight ratio of lungs in the bronchoalveolar lavage fluid; down-regulated the level of pro-inflammatory mediators, including TNF-α, IL-1ß and IL-6; inhibited the phosphorylation of IκB-α, NF-κB p65 and the expression of TLR4, caused by LPS. Taken together, our results suggest that anti-inflammatory effects of magnolol against the LPS-induced acute lung injury may be due to its ability of inhibition TLR4 mediated NF-κB signaling pathways. Magnolol may be a promising potential therapeutic reagent for acute lung injury treatment.

Stevioside suppressed inflammatory cytokine secretion by downregulation of NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 cells.

Stevioside, a diterpene glycoside isolated from Stevia rebaudiana, has been reported to have anti-inflammatory properties. However, the underlying molecular mechanisms are not well understood. The objective of this study was to investigate the molecular mechanism of stevioside in modifying lipopolysaccharide (LPS)-induced signal pathways in RAW264.7 cells. RAW264.7 cells were stimulated with LPS in the presence or absence of stevioside. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) were determined by western blot. The results showed that stevioside dose-dependently inhibited the expression of tumor necrosis factor-α, interleukin-6, and interleukin-1ß in LPS-stimulated RAW264.7 cells. Western blot analysis showed that stevioside suppressed LPS-induced NF-κB activation, IκBa degradation, phosphorylation of ERK, JNK, and P38. Our results suggest that stevioside exerts an anti-inflammatory property by inhibiting the activation of NF-κB and mitogen-activated protein kinase signaling and the release of proinflammatory cytokines. These findings suggest that stevioside may be a therapeutic agent against inflammatory diseases.

A fine balance between CCNL1 and TIMP1 contributes to the development of breast cancer cells.

Cyclin L1 (CCNL1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) are candidate genes involved in several types of cancer. However, the expression of CCNL1 and the relationship between CCNL1 and TIMP1 in breast cancer cells is unknown. Using patients' breast cancer tissues, the expression of CCNL1 and TIMP1 was measured by cDNA microarray and further confirmed by real-time RT-PCR and western blotting. Overexpression or repression of CCNL1 and TIMP1, individually or together, was performed in breast cancer MDA-MB-231 cells by transient transformation methods to investigate their role in breast cancer cell growth. Simultaneously, mRNA and protein expression levels of CCNL1 and TIMP1 were also measured. CCNL1 and TIMP1 expression was significantly elevated in breast cancer tissues compared with that in peri-breast cancer tissues of patients by cDNA microarray and these results were further confirmed by real-time RT-PCR and western blotting. Interestingly, in vitro experiments showed a stimulatory effect of TIMP1 and an inhibitory effect of CCNL1 on growth of MDA-MB-231 cells. Co-expression or co-repression of these two genes did not affect cell growth. Overexpression of CCNL1 and TIMP1 individually induced overexpression of each other. These data demonstrate that there is a fine balance between CCNL1 and TIMP1, which may contribute to breast cancer development.