Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a β-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.     

Mast cell-induced lung injury in mice infected with H5N1 influenza virus

Although an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection both in vivo and in vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.     

Interleukin-22 protects rat PC12 pheochromocytoma cells from serum deprivation-induced cell death

Interleukin-22 (IL-22), an IL-10 family cytokine, mediates the crosstalk between leukocytes and epithelial cells. Previous studies reported that IL-22 expresses in mouse brain, and the rat PC12 cells are responsive to IL-22 stimulation. However, the biological roles of IL-22 in neuronal cells remain largely unknown. We show here that IL-22 activates Stat3, p38 mitogen-activated protein kinases (MAPK), and Akt pathways and inhibits Erk/MAPK pathway in na?ve PC12 cells. We further demonstrate that IL-22 protects na?ve PC12 cells from serum starvation-induced cell death via the Jak1/Stat3 and Akt pathways. We also show that IL-22 has no effects on na?ve PC12 cell proliferation and cannot protect na?ve PC12 cells from 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity. However, IL-22 exerts a dose-dependent protective effect on MPP⁺-induced neurodegeneration in nerve growth factor-differentiated PC12 cells. Overall, our data suggest that IL-22 might play a role in neurological processes. To our knowledge, this is the first report showing that IL-22 confers a neuroprotective function, which may provide a new therapeutic option for treatment of neurodegenerative diseases.     

Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. Dihydrofolate reductase (DHFR) can catalyze 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin (BH4) in the salvage pathway of BH4 synthesis from sepiapterin (SP), a major pigment component contained in the integument of silkworm Bombyx mori mutant lemon (lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkworm B. mori (BmDhfr) and identification of enzymatic properties of BmDHFR. BmDhfr is located on scaffold Bm_199 with a predicted gene model BGIBMGA013340, which encodes a 185-aa polypeptide with a predicted molecular mass of about 21?kDa. Biochemical analyses showed that the recombinant BmDHFR protein exhibited high enzymatic activity and suitable parameters to substrate. Together with our previous studies on SP reductase of B. mori (BmSPR) and the lem mutant, it may be an effective way to industrially extract SP from the lem silkworms in large scale to produce BH4 in vitro by co-expressing BmSPR and BmDHFR and using the extracted SP as a substrate in the future.     

DJ-1 may contribute to metastasis of non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients?? tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (P?=?0.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (P?=?0.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (P?=?0.039). DJ-1 might contribute to the metastasis of NSCLC.     

Effect of surgical castration on expression of TRPM8 in urogenital tract of male rats

Trpm8 (melastatin-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily, encoding a cation channel named TRPM8, has been shown to be a primary androgen-responsive gene and play an important role in prostate physiology. To investigate the expression feature of TRPM8 in urogenital tract of male rats, and whether TRPM8 was also regulated by androgen receptor in these organs, male Sprague–Dawley rats were divided into three groups of 35 animals as follows: sham-operated (SHAM), orchidectomized (ORX), orchidectomized plus DHT treatment (O + D). Organs in urogenital tract, including kidney, prostate, seminal vesicle (SV), testis, epididymis and penis, were collected at different post-castration periods. RT-PCR, real-time PCR and Western blotting were used to detect the expression of androgen receptor (AR) and trpm8 in these tissue. As a result, AR and trpm8 can be detected in all these organs at mRNA or/and protein level. The mRNA expression of trpm8 in kidney, prostate, SV and penis decreased 24 or 72 h after castration and kept decreasing in a time-dependant manner. However, treatment of dihydrotestosterone (DHT) could reverse the effect of surgical castration. Collectively, our data provide evidence that TRPM8 and AR were expressed generally in urogenital tract of male rats, and in these organs, expression of trpm8 was regulated by serum androgen.     

Induction of toll-like receptor 2 positive antigen-presenting cells in spleen of pristane-induced arthritis in rats

Toll-like receptors (TLRs) have been found to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study is to investigate the regulation and potential role of TLR2 in spleen of pristane-induced arthritis (PIA) rat, which can be used to further understand the mechanisms of RA. Arthritis in DA rats was induced by pristane. TLR2 expression in spleen was detected by real-time quantitative PCR and western blotting, and TLR2 expression at both mRNA and protein levels was upregulated in PIA rats. Peptidoglycan (PGN) was systemically administrated to PIA rats, and arthritis severity was evaluated macroscopically and microscopically. Results showed that systemic administration of PGN to PIA rats obviously deteriorated arthritis severity. TLR2 expression on splenocytes and different types of immune cells was measured by flow cytometry. And it was found that TLR2 was mainly expressed on antigen-presenting cells (APCs) of spleen, and the proportion of TLR2⁺ dendritic cells and macrophages in spleen of PIA rats was increased remarkably. Thus, we conclude that the induction of TLR2⁺ APCs in spleen may participate in the maintenance of PIA.     

Life cycle of <Emphasis Type="Italic">Haemaphysalis doenitzi</Emphasis> (Acari: Ixodidae) under laboratory conditions and its phylogeny based on mitochondrial 16S rDNA

The paper investigated the life cycle of the hard tick Haemaphysalis doenitzi under laboratory conditions and its phylogeny based on mitochondrial 16S rDNA. The results revealed that the complete life cycle of H. doenitzi requires a mean duration of 109.6 days ranging from 91 to 137 days and the average prefeeding, feeding and premoulting periods of larvae, nymphs and females and the eggs hatching period are 18.7, 26.9, 38.9, and 25.1 days, respectively. In addition, the weight of engorged females is highly correlated with the number of egg masses laid (r = 0.936, P < 0.001). The female reproductive efficiency index and reproductive fitness index are 13.4 and 12.8, respectively. The mean weight of the engorged nymphs (2.77 mg) moulting to females is much higher than those (1.68 mg) moulting to males, which could be used as an index to predict sexes in this species. The ratio of male to female is 1:1.01. Moreover, multisequence alignments and phylogenetic tree constructed based on the mitochondrial 16S rDNA sequences suggest that H. doenitzi is genetically close to H. longicornis.     

A novel anti-lymphoma protein RE26 from <Emphasis Type="Italic">Rozites emodensis</Emphasis> (Berk.) Moser

A novel antitumor protein, designated RE26, with anti-lymphoma activity was purified from a Tris–HCl buffer extract of Rozites emodensis (Berk.) Moser by three successive steps of ion exchange chromatography. SDS-PAGE and gel filtration chromatography revealed that RE26 is a monomeric protein of 26 kDa, and isoelectrofocusing assay indicated its isoelectric point of 4.3–4.4. RE26 has high stability over a wide pH range (pH 3–11) but is sensitive to temperature and only stable under 40 °C. Partial amino acid sequences of two RE26 peptide fragments were determined by Edman degradation as GLEEEETLLLLFFPP and GTEQE. The half-maximal inhibitory concentration (IC₅₀) of RE26 against tested lymphoma cell lines was around 4 μg/ml. In vitro experiments showed that RE26 could specifically bind to lymphoma cells; activate the caspases, including caspases 3, 8, and 9 in host cells; and induce apoptosis. Experiments in nude mice indicated local RE26 injection adjacent to tumor site could inhibit lymphoma formation.     

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.