Nitric oxide synthase and its function in animal reproduction: an update.

Nitric oxide (NO), a free radical labile gas, is involved in the regulation of various biological functions and physiological processes during animal reproduction. Recently, increasing evidence suggests that the biological role and chemical fate of NO is dependent on dynamic regulation of its biosynthetic enzyme, three distinct nitric oxide synthase (NOS) according to their structure, location and function. The impact of NOS isoforms on reproductive functions need to be timely elucidated. Here, we focus on and the basic background and latest studies on the development, structure, importance inhibitor, location pattern, complex functions. Moreover, we summarize the exactly mechanisms which involved some cell signal pathways in the regulation of NOS with cellular and molecular level in the animal reproduction. Therefore, this growing research area provides the new insight into the important role of NOS male and female reproduction system. It also provides the treatment evidence on targeting NOS of reproductive regulation and diseases.

Update on urine as a biomarker in cancer: a necessary review of an old story.

Introduction: Cancer causes thousands of deaths worldwide each year. Therefore, monitoring of health status and the early diagnosis of cancer using noninvasive assays, such as the analysis of molecular biomarkers in urine, is essential. However, effective biomarkers for early diagnosis of cancer have not been established in many types of cancer.Areas covered: In this review, we discuss recent findings with regard to the use of urine composition as a biomarker in eleven types of cancer. We also highlight the use of urine biomarkers for improving early diagnosis.Expert opinion: Urinary biomarkers have been applied for clinical application of early diagnosis. The main limitation is a lack of integrated approaches for identification of new biomarkers in most cancer. The utilization of urinary biomarker detection will be promoted by improved detection methods and new data from different types of cancers. With the development of precision medicine, urinary biomarkers will play an increasingly important clinical role. Future early diagnosis would benefit from changes in the utilization of urinary biomarkers.

Neural cell adhesion molecule regulates chondrocyte hypertrophy in chondrogenic differentiation and experimental osteoarthritis.

Chondrocyte hypertrophy-like change is an important pathological process of osteoarthritis (OA), but the mechanism remains largely unknown. Neural cell adhesion molecule (NCAM) is highly expressed and involved in the chondrocyte differentiation of mesenchymal stem cells (MSCs). In this study, we found that NCAM deficiency accelerates chondrocyte hypertrophy in articular cartilage and growth plate of OA mice. NCAM deficiency leads to hypertrophic chondrocyte differentiation in both murine MSCs and chondrogenic cells, in which extracellular signal-regulated kinase (ERK) signaling plays an important role. Moreover, NCAM expression is downregulated in an interleukin-1ß-stimulated OA cellular model and monosodium iodoacetate-induced OA rats. Overexpression of NCAM substantially inhibits hypertrophic differentiation in the OA cellular model. In conclusion, NCAM could inhibit hypertrophic chondrocyte differentiation of MSCs by inhibiting ERK signaling and reduce chondrocyte hypertrophy in experimental OA model, suggesting the potential utility of NCAM as a novel therapeutic target for alleviating chondrocyte hypertrophy of OA.

Protective effects of ethyl gallate and pentagalloylglucose, the active components of Qingwen Baidu Decoction, against lipopolysaccharide-induced acute lung injury in rats.

BACKGROUND: The aim of this study was to investigate the bioactive constituents of Qingwen Baidu Decoction (QBD) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Our previous studies showed that ethyl gallate (EG) and pentagalloylglucose (PGG) were the active components of QBD for the treatment of ALI. MATERIALS AND METHODS: We isolated two compounds and identified the structures of them by nuclear magnetic resonance and mass spectrometer. Lung injury was induced by intratracheal instillation with LPS (5 mg/kg). Rats were randomly divided into six groups: Control group; LPS group; 5 mL/kg DEX + LPS group; 6.6 g/kg QBD extract + LPS group; 17.16 mg/kg PGG + LPS group; 7.26 mg/kg EG + LPS group. The effects of compounds on LPS-induced the number of total cells, neutrophils influx, protein leakage, W/D weight ratio and pulmonary histological changes were examined. RESULTS: The results demonstrated that pretreatment with EG and PGG could notably inhibit lung edema and attenuate the pulmonary histological changes (P<0.05). The pretreatment also decreased the number of total cells and polymorphonuclear leukocytes in bronchoalveolar lavage fluid (BALF) (P<0.05). CONCLUSION: Ethyl gallate and pentagalloylglucose of QBD played a protective role in preventing LPS-induced ALI. The results supported further study of EG and PGG as potential candidates for preventing ALI.

NCAM affects directional lamellipodia formation of BMSCs via ß1 integrin signal-mediated cofilin activity.

The neural cell adhesion molecule (NCAM), a key member of the immunoglobulin-like CAM family, was reported to regulate the migration of bone marrow-derived mesenchymal stem cells (BMSCs). However, the detailed cellular behaviors including lamellipodia formation in the initial step of directional migration remain largely unknown. In the present study, we reported that NCAM affects the lamellipodia formation of BMSCs. Using BMSCs from Ncam knockout mice we found that Ncam deficiency significantly impaired the migration and the directional lamellipodia formation of BMSCs. Further studies revealed that Ncam knockout decreased the activity of cofilin, an actin-cleaving protein, which was involved in directional protrusions. To explore the molecular mechanisms involved, we examined protein tyrosine phosphorylation levels in Ncam knockout BMSCs by phosphotyrosine peptide array analyses, and found that the tyrosine phosphorylation level of ß1 integrin, a protein upstream of cofilin, was greatly upregulated in Ncam-deficient BMSCs. Notably, by blocking the function of ß1 integrin with RGD peptide or ROCK inhibitor, the cofilin activity and directional lamellipodia formation of Ncam knockout BMSCs could be rescued. Finally, we found that the effect of NCAM on tyrosine phosphorylation of ß1 integrin was independent of the fibroblast growth factor receptor. These results indicated that NCAM regulates directional lamellipodia formation of BMSCs through ß1 integrin signal-mediated cofilin activity.

Bioactive Components from Qingwen Baidu Decoction against LPS-Induced Acute Lung Injury in Rats.

Qingwen Baidu Decoction (QBD) is an extraordinarily "cold" formula. It was traditionally used to cure epidemic hemorrhagic fever, intestinal typhoid fever, influenza, sepsis and so on. The purpose of this study was to discover relationships between the change of the constituents in different extracts of QBD and the pharmacological effect in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). The study aimed to discover the changes in constituents of different QBD extracts and the pharmacological effects on acute lung injury (ALI) induced by LPS. The results demonstrated that high dose and middle dose of QBD had significantly potent anti-inflammatory effects and reduced pulmonary edema caused by ALI in rats (p < 0.05). To explore the underlying constituents of QBD, we assessed its influence of six different QBD extracts on ALI and analyzed the different constituents in the corresponding HPLC chromatograms by a Principal Component Analysis (PCA) method. The results showed that the pharmacological effect of QBD was related to the polarity of its extracts, and the medium polarity extracts E2 and E5 in particular displayed much better protective effects against ALI than other groups. Moreover, HPLC-DAD-ESI-MSn and PCA analysis showed that verbascoside and angoroside C played a key role in reducing pulmonary edema. In addition, the current study revealed that ethyl gallate, pentagalloylglucose, galloyl paeoniflorin, mudanpioside C and harpagoside can treat ALI mainly by reducing the total cells and infiltration of activated polymorphonuclear leukocytes (PMNs).

[Construction and biological characteristics of H5N1 avian influenza viruses with different patterns of the glycosylation sites in HA protein].

The distribution of glycosylation sites in HA proteins was various among H5 subtype avian influenza viruses (AIVs), however, the role of glycosylation sites to the virus is still unclear. In this study, avian influenza H5N1 viruses with deletion of the glycosylation sites in HA were constructed and rescued by site direct mutation and reverse genetic method, and their biological characteristics and virulence were determined. The result showed that the mutants were confirmed to be corrected by HA gene sequencing and Western blot analysis. The EID50 and TCID50 tested in SPF chick embryo and MDCK cells of a mutant rSdelta158 with deletion of glycosylation site at position 158 were slight lower than that of wild type rescued virus rS, and the plaque diameter of rSdelta158 was significant smaller than that of rS. The EID50 and TCID50 of mutants rSdelta169 and rSdelta290 with deletion of glycosylation sites at position 169 and 290, respectively, were slight higher than that of wild type rescued virus rS, the plaque diameters of rSdelta169 and rSdelta290 were similar as that of rS, but the plaque numbers of rSdelta169 and rSdelta290 were 10-fold higher than that to rS. On the other hand, the rSdelta158, rSdelta169 and rSdelta290 showed similar growth rate in chicken embryo fibroblast as rS. All viruses remained high pathogenicity to SPF chickens. Therefore, the growth of AIV can be affected by changes of glycosylation sites in HA protein, by which the effect is variable in different cells.

[Genome sequencing and phylogenetic analysis of avian influenza viruses subtype H9N2].

Samples of chicken, duck, quail, and pigeon were collected from Jiangsu, Anhui, and Hebei in 2009-2011, and sixteen H9N2 subtype isolates of avian influenza virus (AIV) were identified. The eight full-length genes of 16 AIV isolates were amplified by RT-PCR and sequenced. Genome sequence analysis showed that the amino acid motif of cleavage sites in the HA gene was P-S-R/K-S-S-R, which was consistent with the characterization of the LPAIV, and the Leucine (L) at the amino acid position 226 in the HA genes of all isolates indicated the potential of binding with SAalpha, 2-6 receptor. All isolates had a S to N substitution at residue 31 in the M2 gene, which is related to the resistance phenotype of adamantanes. The key molecular features of 16 AIV isolates from different hosts were same. Genome phylogenetic analysis revealed that all 16 H9N2 subtype AIVs originated from F98-like virus as backbone and formed two new genotypes through reassortment with HA gene of Y280-like virus and PB2 and M genes of G1-like virus. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.

hsBAFF regulates proliferation and response in cultured CD4(+) T lymphocytes by upregulation of intracellular free Ca(2+) homeostasis.

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T-cell activation. Recently, we have demonstrated that treatment of mice with human soluble BAFF (hsBAFF) causes a significant increase of percentages of splenic CD4(+) T lymphocytes dose-dependently, but the CD8(+) T lymphocyte percentages maintained unchanged. Here, we show that hsBAFF significantly enhanced CD4(+) T lymphocyte response of cultured mouse splenic cells, and hsBAFF induced the proliferation and IL-2/IFN-γ secretion of purified CD4(+) T lymphocytes suboptimally stimulated through anti-CD3. Of importance, we observed that IL-2 or IFN-γ cytokine has additive effect on the proliferation and activity of hsBAFF-stimulated CD4(+) T lymphocytes. Using Flow cytometry with fluorescent probe, Fluo-3/AM, we found that hsBAFF elicited [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. This is evidenced by our finding that pretreatment with BAPTA/AM, an intracellular Ca(2+) chelator, significantly attenuated the proliferation of hsBAFF-stimulated CD4(+) T lymphocytes. Subsequently, we revealed that hsBAFF-stimulated CD4(+) T cell proliferation was markedly suppressed after pretreatment with EGTA, an extracellular Ca(2+) chelator, or with 2-APB, an inhibitor of Ca(2+) influx through CRAC channels, respectively, suggesting that extracellular Ca(2+) influx due to hsBAFF is closely associated with [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. In addition, we noticed that hsBAFF-treated cells conferred partial resistance to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor. Taken together, our data indicate that hsBAFF may promote CD4(+) T cell proliferation and response by upregulation of [Ca(2+)](i) homeostasis.

Expression of rabbits BAFF in Escherichia coli with costimulatory effect on B cell proliferation.

In this paper, the cDNA encoding the extracellular soluble domain of rabbit BAFF (rsBAFF) was subcloned into the pET28a vector and the recombinant rsBAFF protein was solubly produced in Escherichia coli. Recombinant rsBAFF protein of 17.1kDa was then purified to a purity of 95% by using Ni(2+)-IDA resin. In vitro, purified rsBAFF protein was able to promote the survival/proliferation of B-lymphocytes of rabbits, mice and humans in the presence of anti-IgM antibodies. These findings indicate that the recombinant rsBAFF produced in E. coli have the bioactivity, and functional cross-reactivity occurs between rabbit and other mammalians BAFF proteins.

Construction and expression of a novel bioactive IFN-alpha2b/CM4 fusion protein in Escherichia coli.

Human interferon alpha2b (IFN-alpha2b) is a pleiotropic cytokine used for the treatment of various cancers. Antibacterial peptide CM4 is a small peptide that can strongly inhibit many kinds of bacteria, fungi, and tumor cells, but it does no harm to normal cells. Here, we describe a protein expression system for the production of IFN-alpha2b/CM4 fusion protein in insoluble form in Escherichia coli, coupled to an efficient dialysis refolding and histidine-tag purification protocol. The expressed IFN-alpha2b/CM4 fusion protein not only displays significantly improved antitumor activity, but also retains the antibacterial-antifungal activity of CM4.

[Construction of recombinant fowlpox virus coexpressing HA gene from H5N1 avian influenza virus and chicken interleukin-2 gene and assessment of its protective efficacy].

The hemagglutinin (HA) gene from H5N1 avian influenza virus and the chicken interleukin 2 (chiIL-2) gene were inserted into a expressing vector p12LS to construct a recombinant transferring vector p12LSH5AIL2, in which HA gene under the control of the promoter Ps was in inverse tandem connection with the chiIL-2 gene under the control of the promoter PE/L. The p12LSH5AIL2 was then used to transfect the chicken embryo fibroblasts (CEF) pre-infected with a wild-type fowlpox virus 282E4 strain, to generate a recombinant fowlpox virus coexpressing the inserted HA and chiIL2 genes (rFPV-H5AIL2). The rFPV-H5AIL2 was obtained and purified by blue plaque screening on the CEF. The in vitro expression of HA gene by rFPV-H5AIL2 was detected in the recombinant fowlpox virus-infected CEFs with an indirect immunofluorescence assay, and the expression of the chiIL2 gene by rFPV-H5AIL2 was confirmed by detection of the chiIL2 mRNA by RT-PCR and by detection of chiIL2 by the indirect immunofluorescence assay. Experiments on SPF and commercial chickens demonstrated that the titer for HI antibodies induced by the rFPV-H5AIL2 was significantly higher than that by the rFPV-HA. The group immunized with the rFPV-H5AIL2 exhibited the similar ratios of protective efficacy and virus shedding as the group immunized with the rFPV-HA in SPF chicken. However, in commercial chicken, the group immunized with the rFPV-H5AIL2 generated significantly higher protection against H5N1 avian influenza virus challenge and lower virus shedding than the group immunized with the rFPV-HA. This study paved the way for further development of a new AIV recombinant vaccine.

Specific IgG activity of bovine immune milk against diarrhea bacteria and its protective effects on pathogen-infected intestinal damages.

Bovine milk antibodies of lactating cows immunized with a multivalent vaccine consisting of whole cells of 17 strains of pathogenic diarrhea bacteria were generated. Using an in vitro mimicked intestine pH environment, we demonstrate that strong activity of their specific IgG may be recovered through the gastrointestinal tract to prevent pathogen-infected intestinal damages, thereby potently inhibiting pathogen-induced diarrhea. This is supported by histological and ultrastructural evidence that specific IgG may effectively abolish pathogen-induced cellular dysfunction of small intestinal mucosa, including tight junctions, brush border, enterocytes, goblet cells, etc. Normal IgG from non-immunized milk is incapable of eliciting the same consequences as specific IgG. Furthermore, we also noticed that specific IgG exerts an effective protection by enhancing splenic natural killer (NK) cell activity in pathogen-infected mice. Our findings indicate that the specific IgG from milk antibodies of immunized lactating bovine may be exploited in therapies for prevention of multibacteria-induced diarrhea.

[Recombinant fowlpox virus coexpressing HA and NA gene from subtype H5N1 of avian influenza virus and its protective efficacy].

The hemagglutinin (HA) and neuraminidase(NA)gene from subtype H5N1 avian influenza virus were directly inserted into the transferring vector pllS, resulting in the recombinant transferring vector p11SH5ANA. Then p11SH5ANA was transfected into the chicken embryo fibroblasts (CEF), which was pre-infected with wild type fowlpox virus, to generate the recombinant fowlpox virus coexpressing H5A and NA (rFPV-11SH5ANA). By selection of blue plaques on the CEF, rFPV-11SH5ANA was obtained and purified. Experiments on SPF chickens demonstrated that the HI antibody titers in chickens vaccinated with HA-NA coexpressed vaccine was higher than those with HA expressed monovalent vaccines, and all chickens receiving either rFPV-11SH5ANA or rFPV-11SH5A were completely protected from the virulent AIV(H5N1) challenge, while those receiving wt-FPV experienced 100% mortality. The results showed that the rFPV-11SH5ANA was a safe and highly efficient gene engineering vaccine candidate for preventing HPAI.

[Influence of nonessential region on protective efficacy of recombinant fowlpox viruses].

Because of the interference of maternal antibodies, the recombinant fowlpox virus (rFPV) vaccine has not been used widely. The selection of a well-defined FPV nonessential region might be a desirable way to solve this problem. Two pairs of primers were designed according to the genome of a pathogenic FPV to amplify two flanking regions (FPV1 and FPV2) of the supposed nonessential region by PCR, and then a series of plasmid vectors were constructed to generate the expression vector pP12LS, which containing FPV1, FPV2, the expression cassette of P11-LacZ reporter gene and the promoter Ps. To abtain the vector pP12LSF, the F gene of ZJ1 strain Newcastle Disease Virus (NDV) was inserted into pP12LS, in which the F gene was located downstream of the promoter Ps. pP12LSF was transfected into chicken embryo fibroblast (CEF) pre-nfected with 282E4 strain FPV. The recombinant FPV, rFPV-FSC, was purified by blue plaque selection. The LacZ and F genes could be expressed by rFPV-FSC after 20 successive passages in CEF. The FPV nonessential region was the only difference between rFPV-FSC and rFPV-FSB. These two rFPVs could induce completely protection in SPF chickens against lethal challenge with F48E8 strain NDV. However, the protective efficacy showed a significant difference in commercial chickens with maternal antibodies. The protective efficacy of rFPV-FSC was 100% and rFPV-FSB was 61.54%. The results indicate that the selection of a well-defined FPV nonessencial region is an effective way to increase the protective efficacy of rFPVs, especially in chickens with maternal antibodies.

[Generation of A/Chicken/Shanghai/F/98 (H9N2) avian influenza virus from eight plasmids].

Eight-plasmid system was used for the generation of Avian influenza virus (AIV) strain A/Chicken/Shanghai/F/98 (H9N2) which was isolated in China in 1998. In this plasmid-based expression system, viral cDNA was inserted beteen the RNA polymerase I (pol I) promoter and terminator sequences. The entire pol I transcription unit was flanked by an RNA polymerase II (pol II) promoter and a poly (A) site. Twenty-four hours after the transfection of eight expression plasmid into cos1 cells, the supernatant and cos1 cells transfected were inoculated into the allantoic cavity of 10-day-old specific-pothgen-free (SPF) chicken eggs. The HA titer was determined after passage of the rescued virus in chicken eggs, and as high as that of the parental wild-type virus.

Suppression of IL-8 gene transcription by resveratrol in phorbol ester treated human monocytic cells.

Resveratrol (3,4',5-trihydroxy-trans-silbene), a natural phytoalexin found in grapes and other food products, has promising anti-inflammatory and anticancer effects. To observe the modulation of interleukin-8 (IL-8) production in human monocytic cells by resveratrol and explore its mechanism at the gene transcription level, U937 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24h. IL-8 protein in supernatants was measured by radioimmunoassay. The cytotoxicity of PMA, dexamethasone and resveratrol was accessed by MTT cell proliferation assay. The RNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and IL-8 were detected by RT-PCR using specific primers. DNA binding activities of NF-kappaB and AP-1 were examined by electrophoretic mobility shift assay (EMSA). 0.01-100 nM PMA could significantly induce IL-8 production in U937 cells; 10 microM Dexamethasone and 10, 1, 0.1 microM resveratrol could inhibit PMA-induced IL-8 protein production and mRNA accumulation. The cytotoxicity did not contribute to their inhibitory effect. The DNA binding activity of AP-1 was inhibited by dexamethasone and resveratrol, but resveratrol has little effect on PMA-induced NF-kappaB activation. Resveratrol could inhibit PMA-induced IL-8 production in U937 cells at protein and mRNA levels. The suppression of IL-8 gene transcription by resveratrol was, at least partly, due to inhibition of AP-1 activation.