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Dimethyl sulfide (DMS) is an important and dominant trace gas that is transferred from the ocean to the atmosphere; however, the production of DMS from marine algae is still rudimentary, with the enzymatic cleavage of dimethylsulfoniopropionate (DMSP) to DMS and acrylic acid (AA), one of the main considered pathways. Dissolved DMSP (DMSPd) was supplied to batch cultures of Prorocentrum minimum at different algal growth stages and the kinetics of overall DMSPd consumption and DMS (AA) production were investigated. The kinetics of overall DMSPd consumption and DMS (AA) production from DMSPd were different during the growth cycle. The cultures displayed Michaelis–Menten-type kinetics for DMS production during the entire growth period, which yielded an apparent half-saturation constant (Km(app)) value of 37.8 ± 12.8 μM and a maximum rate (Vmax) value of 75.6 ± 12.2 nmol min−1 (1010 cells)−1. The production rates of AA had a nearly linear fit over the concentration range of the added DMSPd except during mid-exponential growth phase. A Km(app) value of 1.73 ± 0.75 μM and a Vmax value of 2.36 ± 0.12 μmol min−1 (1010 cells)−1 were observed. The ratio of DMS/AA increased with the concentration of the added DMSPd, which was less than 12% during the entire growth period. The low ratios of DMS/(DMSP + AA), which ranged from 0.01 to 1.83%, demonstrate that degradation of DMSP tends to take a non-DMS-production pathway. The ratios of AA/(DMSP + AA) varied from 9 to 95% during the stationary phase and from 6 to 69% during the senescent phase and declined with substrate concentration when different concentrations of DMSPd were added. The degradation ratios of DMSPd were related to the initial concentrations of DMSPd.

The spatial distributions, fluxes, and environmental effects of non-methane hydrocarbons (NMHCs) were investigated in the Yellow Sea (YS) and the East China Sea (ECS) in spring. The average concentrations of ethane, propane, i-/n-butane, ethylene, propylene and isoprene in the seawater were 18.1 ± 6.4, 15.4 ± 4.7, 6.8 ± 2.9, 6.4 ± 3.2, 67.1 ± 26.7, 20.5 ± 8.7 and 17.1 ± 11.1 pmol L−1, respectively. The alkenes in the surface seawater were more abundant than their saturated homologs and NMHCs concentrations (with the exception of isoprene) decreased with carbon number. The spatial variations of isoprene were consistent with the distributions of chlorophyll a (Chl-a) and Chaetoceros, Skeletonema, Nitzschia mainly contributed to the production of isoprene, while the others’ distributions might be related to their photochemical production. Observations in atmospheric NMHCs indicated alkanes in the marine atmosphere decreased from inshore to offshore due to influence of the continental emissions, while alkenes were largely derived from the oceanic source. In addition, no apparent diurnal discrepancy of atmospheric NMHCs (except for isoprene) were found between daytime and night. As the main sink of NMHCs in seawater, the average sea-to-air fluxes of ethane, propane, i-/n-butane, ethylene and propylene were 31.70, 29.75, 18.49, 15.89, 239.6, 67.94 and 52.41 nmol m−2 d−1, respectively. The average annual emissions of isoprene accounted for 0.1–1.3% of the global ocean emissions, which indicated that the coastal and shelf areas might be significant sources of isoprene. Furthermore, this study represents the first effort to estimate the environmental effects caused by NMHCs over the YS and the ECS and the results demonstrated contributions of alkanes to ozone and secondary organic aerosol (SOA) formation were lower than those of the alkenes and the largest contributor was isoprene.

Dimethylsulfoniopropionate (DMSP) is widespread in the oceans, and its biological metabolite, dimethyl sulfide (DMS), plays an important role in the atmosphere. The Antarctic region has become a hotspot in DMS studies due to the high spatial and temporal variability in DMS(P) concentration, but the level of bacterial DMS production remains unclear. In this study, a bacterium isolated from Antarctic floating ice, Rhodococcus sp. NJ-530, was found to metabolize DMSP into DMS, and the rate of DMS production was measured as 3.96 pmol·mg protein-1 ·h-1 . Rhodococcus sp. NJ-530 had a DddD-Rh enzyme containing two CaiB domains, which belonged to the CoA-transferase III superfamily. However, the DddD-Rh had a molecular weight of 73.21 kDa, which was very different from previously characterized DddD enzymes in sequence and evolution. In vitro assays showed that DddD-Rh was functional in the presence of acetyl-CoA. This was the first functional DddD from Gram-positive Actinobacteria. Moreover, a quantitative real-time polymerase chain reaction revealed that high temperature facilitated the expression of dddD-Rh, and changes of salinity had little effect on it. This study adds new evidence to the bacterial DMS production in the Southern Ocean and provides a basis for investigating the metabolic mechanism of DMSP in extreme environments.

A mesocosm experiment was conducted in Wuyuan Bay (Xiamen), China, to investigate the effects of elevated p CO2 on the phytoplankton species Phaeodactylum tricornutum (P. tricornutum), Thalassiosira weissflogii (T. weissflogii) and Emiliania huxleyi (E. huxleyi) and their production ability of dimethylsulfide (DMS), dimethylsulfoniopropionate (DMSP), as well as four halocarbon compounds, bromodichloromethane ( CHBrCl2 ), methyl bromide ( CH3Br ), dibromomethane ( CH2Br2 ) and iodomethane ( CH3I ). Over a period of 5 weeks, P. tricornuntum outcompeted T. weissflogii and E. huxleyi, comprising more than 99 % of the final biomass. During the logarithmic growth phase (phase I), mean DMS concentration in high p CO2 mesocosms (1000 µ atm) was 28 % lower than that in low p CO2 mesocosms (400 µ atm). Elevated p CO2 led to a delay in DMSP-consuming bacteria concentrations attached to T. weissflogii and P. tricornutum and finally resulted in the delay of DMS concentration in the high p CO2 treatment. Unlike DMS, the elevated p CO2 did not affect DMSP production ability of T. weissflogii or P. tricornuntum throughout the 5-week culture. A positive relationship was detected between CH3I and T. weissflogii and P. tricornuntum during the experiment, and there was a 40 % reduction in mean CH3I concentration in the high p CO2 mesocosms. CHBrCl2 , CH3Br , and CH2Br2 concentrations did not increase with elevated chlorophyll a (Chl a) concentrations compared with DMS(P) and CH3I , and there were no major peaks both in the high p CO2 or low p CO2 mesocosms. In addition, no effect of elevated p CO2 was identified for any of the three bromocarbons.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

The spatial distributions of dimethylsulfide (DMS), dimethylsulfoniopropionate (DMSP), DMSP-lyase activity (DLA) and their controlling factors including nutrients, phytoplankton community and bacterial abundance were investigated in the East China Sea (ECS) during fall from October 19 to November 2, 2015. Diatoms and dinoflagellates dominated the phytoplankton community, while other taxonomic groups were rare and mainly found in the oligotrophic open sea. Affected by the high nutrients concentrations, Chl a, DMS, DMSP and DLA showed high values in eutrophic inshore waters, and decreased from the costal zones to the open sea. Statistical analysis suggested that diatoms and dinoflagellates were the main controlling factors of DMS, DMSP, and DLA in ECS. For size-fractionated samples, a reduced contribution of the microplankton from inshore stations to offshore stations affected by the trophic conditions was noted. Meanwhile, this decrease in microplankton led to an increase in the ratio of DLA contributed by picoplankton and free-living bacteria from the estuary area to the offshore region. The DMS sea-to-air flux was calculated using the equation of Nightingale et al. (Glob Biogeochem Cy 14(1):373–387, 2000), and approximately 2.88 × 10−2 Tg of sulfur was transferred from the sea into the atmosphere in the form of DMS in ECS during fall.

Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy.

Distributions and variations of biogenic sulfur compounds including dimethylsulfide (DMS), dissolved and total dimethylsulfoniopropionate (DMSPd and DMSPt) and acrylic acid (AA) were investigated in coastal waters off Qingdao, China during the late-bloom and after-bloom periods of the Ulva prolifera bloom of 2015. DMSPd, DMS and AA concentrations after the bloom were significantly higher than during the late-bloom, but DMSPt concentrations in surface waters began to decrease. High concentrations of these compounds in the surface layer were associated with the bloom, with the exception of increased concentrations of DMSPt in the middle layer as decaying U. prolifera debris settled. The sea-to-air fluxes of DMS were estimated to be 18.08 and 24.24 μmol m−2 d−1 during the late-bloom and after-bloom, and about three times higher than the reported average fluxes of the Yellow Sea, which highlighted the impacts of U. prolifera blooms on DMS emissions.

Dimethylsulfide (DMS), a climatically important gas generated by dimethylsulfoniopropionate (DMSP) degradation, plays an important role in the global sulfur cycle and affects the global climate. Marine bacteria are the primary mediators of DMSP degradation and DMS production. Here, we present the complete genome sequence of Rhodococcus sp. NJ-530, isolated from Antarctic sea ice, which utilizes DMSP as a sole carbon and energy source, degrading DMSP into DMS. The genome of strain NJ-530 consists of 7371 protein-coding sequences (CDSs) with 54 tRNA genes and 15 rRNA operons as 5S-16S-23S rRNA. The strain has one circular chromosome of 6,408,544 bp with 6331 CDSs and 62.41% GC content. Genomic annotation revealed that Rhodococcus sp. NJ-530 may have a DMSP cleavage gene cluster, including dddD, dddB and dddC, suggesting the existence of the DddD-type DMSP cleavage pathway. The complete genome sequence of Rhodococcus sp. NJ-530 will provide useful information for better understanding of the molecular mechanism underlying marine DMSP degradation and Antarctic DMS production.

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal women's health.

Since 2007, the green algal bloom occurred along the coast of Qingdao city every summer. In this study, we focused on how the carbonate system responded to the green algal bloom. Dissolved inorganic carbon (DIC), total alkalinity (TA) and pH were measured in two cruises during the green algal post-bloom period in summer 2008 in the southern Yellow Sea. Data showed that the average values of alkalinity calculated from DIC and pH were 2217 and 2241 μmol kg−1 during the first and second cruises, respectively. The alkalinity measured by Gran titration, however, was abnormal, with average values of 3333 and 3280 μmol kg−1. The abnormal measured TA values were probably due to both direct contribution of organic matter and its side reaction in determining TA using Gran titration. Another significant phenomenon was an increase of DIC and a decrease of pH after the bloom. The changes of DIC and pH during the two cruises were +87 μmol kg−1 and −0.18, respectively. Normalized DIC and TA showed that DIC was not conserved after the bloom while TA could be still conserved. In contrast to green algal bloom period, during the post-bloom period, pCO2 values were significantly raised in seawater, with average values of 596 and 981 μatm, resulting in CO2 outgassing from seawater. These results showed that carbonate system in seawater could be greatly altered by green algal bloom.

Context Augmented uterine artery (UA) production of vasodilators, including nitric oxide (NO) and hydrogen sulfide (H2S), has been implicated in pregnancy-associated and agonist-stimulated rise in uterine blood flow that is rate-limiting to pregnancy health. Objective Developing a human UA endothelial cell (hUAEC) culture model from main UAs of nonpregnant (NP) and pregnant (P) women for testing a hypothesis that pregnancy augments endothelial NO and H2S production and endothelial reactivity to vascular endothelial growth factor (VEGF). Design Main UAs from NP and P women were used for developing hUAEC culture models. Comparisons were made between NP- and P-hUAECs in in vitro angiogenesis, activation of cell signaling, expression of endothelial NO synthase (eNOS) and H2S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase, and NO/H2S production upon VEGF stimulation. Results NP- and P-hUAECs displayed a typical cobblestone-like shape in culture and acetylated low-density lipoprotein uptake, stained positively for endothelial and negatively for smooth muscle markers, maintained key signaling proteins during passage, and had statistically significant greater eNOS and CBS proteins in P- vs NP-hUAECs. Treatment with VEGF stimulated in vitro angiogenesis and eNOS protein and NO production only in P-hUEACs and more robust cell signaling in P- vs NP-hUAECs. VEGF stimulated CBS protein expression, accounting for VEGF-stimulated H2S production in hUAECs. Conclusion Comparisons between NP- and P-hUAECs reveal that pregnancy augments VEGF-stimulated in vitro angiogenesis and NO/H2S production in hUAECs, showing that the newly established hUAEC model provides a critical in vitro tool for understanding human uterine hemodynamics.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) mediates vascular endothelial growth factor (VEGF)-stimulated endothelial cytoskeleton remodeling and migration; however, the underlying mechanisms are elusive. Covalent adduction of a NO moiety (NO(•)) to cysteines called S-nitrosylation (SNO) is a key NO signaling pathway. The small actin-binding protein cofilin-1 (CFL1) is essential for actin cytoskeleton remodeling. We investigated whether S-nitrosylation regulates CFL1 function and endothelial cytoskeleton remodeling and migration upon VEGF stimulation. VEGF rapidly stimulated S-nitrosylation of CFL1, which was blocked by NO Synthase inhibition and eNOS knockdown by specific eNOS-siRNA. Cys80 and Cys139 were identified as the major SNO-sites in CFL1 by LC-MS/MS. The actin severing activity of recombinant SNO-mimetic CFL1 (C80/139A DMA-CFL1), but not SNO-deficient CFL1 (C80/139S DMS-CFL1), was significantly greater than that of wild-type CFL1 (wt-CFL1). When wt-CFL1 and its mutants were overexpressed in endothelial cells, basal actin bound wt-CFL1 was undetectable but significantly increased by VEGF; basal actin bound DMA-CFL1 was readily high and basal actin bound DMS-CFL1 was detectable but low, and both were unresponsive to VEGF. Treatment with VEGF significantly increased filamentous (F-) actin and filopodium formation and cell migration in endothelial cells. Overexpression of wt-CFL1 inhibited VEGF-induced F-actin formation. Overexpression of DMA but not DMS CFL1 decreased basal but not VEGF-stimulated F-actin formation. Overexpression of DMA but not DMS CFL1 suppressed VEGF-stimulated filopodium formation and migration in endothelial cells. Thus, S-nitrosylation of CFL1 provides a novel signaling pathway post-NO biosynthesis via eNOS-derived NO for endothelial cytoskeleton remodeling and migration upon VEGF stimulation.

Adduction of a nitric oxide (NO) moiety (NO•) to cysteines termed as S-nitrosylation (SNO) has emerged as a crucial mechanism for NO signaling crucial for mediating the vascular effects of estrogens. Mitochondrion is a known vascular risk factor; however, the effects of estrogens on mitochondrial SNO are incompletely understood. In this study we determined the effects of estradiol-17β (E2β) on mitochondrial protein SNO in primary human umbilical vein endothelial cells and compared the mitochondrial nitroso-proteomes in E2β- and a NO donor S-nitrosoglutathione (GSNO)-treated cells using a proteomics approach. Treatment with 10 nM E2β and 1 mM GSNO for 30 minutes significantly increased the levels of mitochondrial SNO-proteins. Subcellular localization of SNO-proteins showed mitochondria as the major cellular organelle for protein SNO in response to E2β and GSNO. E2β stimulated mitochondrial endothelial nitric oxide synthase (eNOS) phosphorylation and mitochondrial protein SNO that was enhanced by overexpression of mitochondrion or Golgi, but not membrane targeting eNOS constructs. We identified 11, 32, and 54 SNO-proteins in the mitochondria from the untreated, E2β-, and GSNO-treated human umbilical vein endothelial cells, respectively. Comparisons of the nitroso-proteomes revealed that common and different mitochondrial SNO-proteins were affected by endogenous NO on E2β stimulation and exogenous NO from donor. These SNO-proteins were associated with various mitochondrial functions, including energy and redox regulation, transport, iron homeostasis, translation, mitochondrial morphology, and apoptosis, etc. Collectively, we conclude that estrogens rapidly stimulate protein SNO in endothelial mitochondria via mitochondrial eNOS, providing a mechanism for mediating the vascular effects of estrogens.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between “open” and “closed” states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction.

Spatial distribution and sea-to-air flux of isoprene in the East China Sea and the South Yellow Sea in July 2013 were investigated. This study is the first to report the concentrations of isoprene in the China marginal seas. Isoprene concentrations in the surface seawater during summer ranged from 32.46 to 173.5 pM, with an average of 83.62 ± 29.22 pM. Distribution of isoprene in the study area was influenced by the diluted water from the Yangtze River, which stimulated higher in-situ phytoplankton production of isoprene rather than direct freshwater input. Variations in isoprene concentrations were found to be diurnal, with high values observed during daytime. A significant correlation was observed between isoprene and chlorophyll a in the study area. Relatively higher isoprene concentrations were recorded at stations where the phytoplankton biomass was dominated by Chaetoceros, Skeletonema, Pennate-nitzschia, and Thalassiosira. Positive correlation was observed between isoprene and methyl iodide. In addition, sea-to-air fluxes of isoprene approximately ranged from 22.17 nmol m−2 d−1–537.2 nmol m−2 d−1, with an average of 161.5 ± 133.3 nmol m−2 d−1. These results indicate that the coastal and shelf areas may be important sources of atmospheric isoprene.

Canidae fecal flora reflects the distal gut microbial community structure. Cuon alpinus was captured on Qilian Mountain in Gansu province of China, and other animals, including Canis lupus, Vulpes vulpes, Vulpes corsac and Nyctereutes procyonoides, were captured at the nearby Dalai Lake Nature Reserve in Inner Mongolia. Fecal samples were collected 2 h after defecation from five healthy unrelated adult animals of each species. None of the animals had recently fed or been administered any drugs or additives that could influence the composition or diversity of the fecal flora. Using traditional culture-dependent methods, we investigated the composition and diversity of the fecal flora of these five Canidae species. Gram-positive spore bacilli, gram-positive having no spore bacilli, gram-positive coccobacteria, gram-negative coccobacteria and gram-negative bacilli were first identified through microscopic observation and then formal identification tests were carried out, including oxygen needing, methyl red and Acetyl methyl methanol test, catalase, gelatin liquefaction, KNO3 reduction, indole, fermentation of saccharides and mellows, sodium citrate and NaCl-phily and so on. Based on the results of these physiological and biochemical tests, along with the morphological description, species from approximately 19–21 genera were identified in the feces. The number of genera in the feces was 22 in C. lupus, 19 in C. alpinus, 21 in N. procyonoides, 21 in V. corsac and 19 in V. vulpes, as well as some unidentified strains. Although, some strains were endemic to the Canidae gut, there were some differences in the community among individuals and species. The Canidae fecal flora comprised 1010–1011 colony forming units/g of feces (wet weight). The amount of bacteria reached 1.442 × 1011 cfu/g in C. lupus, 8.330 × 1010 cfu/g in V. vulpes, 8.170 × 1010 cfu/g in V. corsac, 8.620 × 1010 cfu/g in N. procyonoides and 1.485 × 1011 cfu/g in C. alpinus. The amount of bacteria was significantly different among species (P

The complete mitochondrial genome sequence of the raccoon dog (Nyctereutes procyonoides) was determined by using the long and accurate polymerase chain reaction. The entire mitochondrial genome sequence is 16,713 bp in length contains two ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and 1 control region. Most mitochondrial genes are encoded on the H strand, except for the ND6 gene and 8 tRNA genes. The base compositions of mitochondrial genomes present clearly A–T skew. All the transfer RNA genes can be folded into the typical cloverleaf-shaped structure except tRNA-Ser (AGY), which lacks the dihydrouridine arm. Protein-coding genes mainly initiate with ATG and terminate with TAA. Some reading frame intervals and overlaps are found in the mitochondrial genome. The control region can be divided into three domains: the extended termination associated sequences (ETASs) domain, the central conserved domain and the conserved sequence blocks (CSBs) domain. Three conserved sequence blocks (CSBs) and one extended termination associated sequences (ETAS-1) is found in the control region. The phylogenetic analysis based on the concatenated data set of 14 genes in the mitochondrial genome of Canidae shows that the raccoon dog has close phylogenetic position with the red fox (Vulpes vulpes) and they constitute a clade which has an equil evolutionary position with the clade formed by the genera Canis and Cuon.

Placental angiogenesis contributes to the pathogenesis of preeclampsia (PE) that affects 5-8% of all human pregnancies. MicroRNA (miRNA) are a class of noncoding 21- to 25-nucleotide RNA that negatively regulate gene expression posttranscriptionly.The aim of this study was to test the hypothesis that miRNA are differentially expressed in healthy term and PE placentas and a subclass of angiogenesis-associated miRNA are increased by PE.Total miRNA were extracted from villous placental tissues from healthy term and severe preeclamptic pregnancies. Differential miRNA expression was analyzed by microarray and real-time quantitative PCR. Angiogenesis-associated miRNA were analyzed by target prediction databases. In situ hybridization was used to localize miRNA. Target verification was performed by transfection of miRNA precursors or antagomirs into endothelial and BeWo cells and luciferase reporter assays.Three highly expressed miRNA (miR-17, -20a, and -20b) were found significantly increased in PE compared with healthy term placentas (n = 10 per group). They target on the same group of genes important for angiogenesis. miR-20b was expressed primarily in villous syncytiotrophoblasts in term placenta. Overexpression or inhibition of miR-20b differentially regulated mRNA expression of those genes in endothelial vs. trophoblast cells. Luciferase reporter assay showed that miR-20b targets EPHB4 and ephrin-B2 that have been shown to be critical for early human placental development. Placental ephrin-B2 mRNA was significantly down-regulated in PE compared with normotensive pregnancies.miR-17, miR-20a, and miR-20b are differentially regulated in human placentas by PE. They regulate EPHB4 and ephrin-B2 expression in trophoblast and endothelial cells via the same "seed" sequence, suggesting their roles in early placental development.

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.

Olfactory receptors (ORs), the first dedicated molecules with which odorants physically interact to arouse an olfactory sensation, constitute the largest gene family in vertebrates. Dogs and wolves, like many other mammals, have a highly developed capability to detect and identify odorant molecules, even at minimum concentrations. In this study, the olfactory receptor repertoire from domestic dog and its closest relative, the wolf, were sequenced to estimate the fraction of pseudogenes in each subspecies. The fraction of disrupted olfactory receptor genes in dog was 17.78%, whereas, that in wolf was 12.08%. As expected the dog was less dependent on olfaction than the wolf, and the dog had more olfactory receptor pseudogenes. However, the observed difference between the two subspecies was not at the significant level (χ 2 = 1.388, p = 0.239 > 0.05). The values indicated that although domestication might play a role in the reduction of OR genes, it could not be concluded that the living environment provided by domestication lead to a significant reduction of the functional olfactory receptor repertoire. Furthermore, the purpose of domestication may also have influence on the ratio of functional olfactory receptor genes reduction.

Nitric oxide (NO) plays a key role in placental biology, and placental dysfunction is the main pathogenesis pathway for preeclampsia, yet the direct placental targets of NO actions have not been determined. Covalent adduction of an NO moiety to cysteines, termed S-nitrosylation (SNO), is emerging as a key route by which NO can directly modulate protein functions. This study was conducted to analyze global S-nitroso (SNO)-proteins in human placentas and to determine if their levels differ in normotensive versus severe preeclamptic placentas. Although total nitrite/nitrate increased, total levels of SNO-proteins and nitrosylated forms of endothelial NO synthase and heat shock protein 90 were decreased by preeclampsia. We further compared normotensive and preeclamptic placental nitroso-proteomes (total SNO-protein profiles) by using a biotin and CyDye switch test combined with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and identified SNO-proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Numerous SNO-proteins were displayed as spots on 2D-DIGE gels. One hundred spots of interest were excised; 46 spots were identified, of which 8 spots were novel SNO-proteins; levels of 15 spots were increased, and 6 spots were decreased, and the rest were unchanged by preeclampsia. Pathway analysis suggested that placental SNO-proteins are involved in regulating various cellular functions including protein synthesis, cell movement and metabolism, cell signaling, and other functions. These data therefore show for the first time that SNO is a crucial mechanism by which NO directly regulates placental proteins linked to various biological pathways. The significantly altered placental nitroso-proteome in preeclampsia suggests that SNO plays a role in the placental pathophysiology in preeclampsia.

Bored pile as a major type of foundation, during the construction process, hole collapse or weak air tightness of catheter leads mud layers or broken pile, which makes bearing capacity reduction or even complete loss leading differential settlement of building foundations. In this paper, two-dimensional simulation of the pile has been made by finite element analysis software ADINA. By using of large-scale numerical analysis software MATLAB, the data obtained has been fitted deriving empirical formulas, which researches how different thickness and depth of mud layers impact on bearing capacity of bored piles, having a certain engineering applications.

Understanding basic reproductive physiology is crucial for the management of both captive and free-ranging wolf. In the present study, we determined hormonal changes during pregnancy and the estrous cycle in captive female wolf by measuring fecal steroids collected during the breeding season with high performance liquid chromatography (HPLC). These biochemical analyses were validated using chemical devivatization and mass spectrometry, and interpreted along with the behavioral data. All four females undergoing estrus cycles were copulated with their partners and delivered pups successfully. We found that estradiol concentrations were significantly higher during the estrus cycle than other stages (p

The production of dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) by marine microalgae was investigated to elucidate more on the role of marine phytoplankton in ocean-atmosphere interactions in the global biogeochemical sulfur cycle. Axenic laboratory cultures of four marine microalgae-Isochrysis galbana 8701, Pavlova viridis, Platymonas sp. and Chlorella were tested for DMSP production and conversion into DMS. Among these four microalgae, Isochrysis galbana 8701 and Pavlova viridis are two species of Haptophyta, while Chlorella and Platymonas sp. belong to Chlorophyta. The results demonstrate that the four algae can produce various amounts of DMS(P), and their DMS(P) production was species specific. With similar cell size, more DMS was released by Haptophyta than that by Chlorophyta. DMS and dissolved DMSP (DMSPd) concentrations in algal cultures varied significantly during their life cycles. The highest release of DMS appeared in the senescent period for all the four algae. Variations in DMSP concentrations were in strong compliance with variations in algal cell densities during the growing period. A highly significant correlation was observed between the DMS and DMSPd concentrations in algal cultures, and there was a time lag for the variation trend of the DMS concentrations as compared with that of the DMSPd. The consistency of variation patterns of DMS and DMSPd implies that the DMSPd produced by phytoplankton cells has a marked effect on the production of DMS. In the present study, the authors’ results specify the significant contribution of the marine phytoplankton to DMS(P) production and the importance of biological control of DMS concentrations in oceanic water.

The whole mitochondrial genome sequence of red fox (Vuples vuples) was determined. It had a total length of 16 723 bp. As in most mammal mitochondrial genome, it contained 13 protein coding genes, two ribosome RNA genes, 22 transfer RNA genes and one control region. The base composition was 31.3% A, 26.1% C, 14.8% G and 27.8% T, respectively. The codon usage of red fox, arctic fox, gray wolf, domestic dog and coyote followed the same pattern except for an unusual ATT start codon, which initiates the NADH dehydrogenase subunit 3 gene in the red fox. A long tandem repeat rich in AC was found between conserved sequence block 1 and 2 in the control region. In order to confirm the phylogenetic relationships of red fox to other canids, phylogenetic trees were reconstructed by neighbor-joining and maximum parsimony methods using 12 concatenated heavy-strand protein-coding genes. The result indicated that arctic fox was the sister group of red fox and they both belong to the red fox-like clade in family Canidae, while gray wolf, domestic dog and coyote belong to wolf-like clade. The result was in accordance with existing phylogenetic results.

The diets of the wolf Canis lupus, red fox Vulpes vulpes and raccoon dog Nyctereutes ussurienusis Matschie were studied in the grassland of eastern Inner Mongolia from August to October in 2007 and from March to July in 2008. Feces were collected from dens and latrines. The frequency of occurrence and the dry weight of the remains of each food item in the feces were calculated. Livestock (50.65% frequency of occurrence) constituted the bulk of the wolf diet, while small rodents (76.31% frequency of occurrence) were most frequently eaten by the foxes. Small rodents (22.69% frequency of occurrence) were important to the raccoon dogs but they also frequently consumed birds (39.81% frequency of occurrence) and insects (26.39% frequency of occurrence). The wolf was the most omnivorous and the red fox was the least omnivorous of these three kinds of canidae according to the diversity index. The food niches of the wolf and the red fox overlapped more than that of the raccoon dog and other species. These three kinds of canidae shared many resources and consequently competition occurred to them. However, their diets also differed to some extent which helped them to avoid competition. Furthermore, raccoon dogs were dormant in winter when food was scarce, which may be the reason why all these species could coexist in a rather unproductive grassland.

On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

A fibroblast line from ear marginal tissue of Luxi cattle (LXCEM2/2) was successfully established by direct culturing of explants. Biological analysis showed that the population doubling time (PDT) for reviving cells was approximately 24h. Measurement of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) isoenzymes showed no cross-contamination among the cells. Karyotyping showed that the frequency of cells with chromosome number 2n=60 was 90.7-92.2%. Tests for bacteria, fungi, viruses and mycoplasma were negative. The efficiencies of expression of pEGFP-N3, pEYFP-N1 and pDsRed1-N1 were between 6.3% and 31.6% at 24h, 48h and 72h after transfer; at 24h, fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. Every index of the Luxi cattle cell line meets the quality control standards of the American Type Culture Collection (ATCC). Not only has the germline of this important cattle breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

The full-length of the cDNA sequence of Beijing fatty chicken ADSL gene was investigated by RT-PCR and RACE in this study. The results demonstrated that the complete ADSL cDNA revealed an open reading frame of 1455 nucleotides coding for 485 amino acid residues. The promoter of chicken ADSL cDNA showed typical features of house-keeping genes: no canonical TATA and CAAT boxes, 72.65% GC in 234 bp near the start codon, and there was a C-28T mutation, which caused the site CTCC mutating to the NRF-2 binding site CTTC. The complete open reading frame of the ADSL gene was inserted into expression vector pGEX-4T-1 and the fusion expression vector pGEX-ADSL was constructed. The pGEX-ADSL was transformed into Escherichia coli BL21 (DE3), positive cloning screened, induced and expresses by IPTG. The SDS-PAGE electrophoresis results showed that there were specific bands, about 80.5 kD and an isoelectric point of 6.79, which reached 26.9% of total cell proteins induced in 5hrs, and was an insoluble inclusion body. Under optimal conditions, ADSL was purified by Glutathione Sepharase 4B affinity chromatography, and Western blotting analysis showed that the fused protein was ADSL. This research was a foundation for further research into ADSL’s biological function and its application identification.

Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNO(Cys80) of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling.

Beta-arrestins (β-arrs) are initially known as negative regulators of G protein-coupled receptors (GPCRs). Recently, there is increasing evidence suggesting that β-arrs also serve as scaffolds and adapters that mediate distinct intracellular signal transduction initiated by GPCR activation. In the previous study, we have shown that metabotropic glutamate receptor 7 (mGluR7) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling may be involved in the developmental sevoflurane neurotoxicity. In the present study, we showed that activation of mGluR7 with a group III mGluRs orthosteric agonist LAP4 or an atypical mGluR7 allosteric agonist N,N'-bis(diphenylmethyl)-1,2-ethanediamine dihydrochloride (AMN082) significantly attenuated sevoflurane-induced neuronal apoptosis. Interestingly, this neuroprotective role of LAP4 could be partially reduced by β-arr1 small interfering RNA (siRNA) or β-arr2 siRNA transfection. In contrast, β-arr2 siRNA transfection alone abolished the effects of AMN082 on sevoflurane neurotoxicity. In addition, administration of LAP4 or AMN082 significantly enhanced Phospho-ERK1/2 in sevoflurane neurotoxicity, which could be abrogated by β-arr2 siRNA transfection, but not by β-arr1 siRNA transfection. Increased β-arr2-dependent Phospho-ERK1/2 signaling alleviated sevoflurane neurotoxicity by inhibiting bad phosphorylation. We also found that the neuroprotective role of AMN082 was completely reversed by ERK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126). Alternatively, treatment with U0126 partially suppressed the neuroprotective of LAP4, suggesting that other mechanisms may be implicated in this process. Further investigation indicated that, in the scenario of sevoflurane neurotoxicity, application of LAP4 (but not AMN082) increased the interaction of β-arrs with transcriptional factors CREB binding protein (CBP) and p300. LAP4 also enhanced the β-arr1-dependent H3 and H4 acetylation in sevoflurane neurotoxicity. For the behavior study, treatment with LAP4 or AMN082 significantly improved the emotional and spatial learning and memory disorders induced by postnatal sevoflurane exposure. These results suggested that β-arr1 and 2 may differently modulate mGluR7 signaling in developmental sevoflurane neurotoxicity. This study also reveals a β-arr-biased agonism at GPCRs (e.g. mGluR7).

Abnormal histone acetylation occurs during neuropathic pain through an epigenetic mechanism. Silent information regulator 1 (sir2 or SIRT1), a NAD-dependent deacetylase, plays complex systemic roles in a variety of processes through deacetylating acetylated histone and other specific substrates. But the role of SIRT1 in neuropathic pain is not well established yet. The present study was intended to detect SIRT1 content and activity, nicotinamide (NAM) and nicotinamide adenine dinucleotide (NAD) in the spinal cord using immunoblotting or mass spectroscopy over time in mice following chronic constriction injury (CCI) or sham surgery. In addition, the effect of intrathecal injection of NAD or resveratrol on thermal hyperalgesia and mechanical allodynia was evaluated in CCI mice. Finally, we investigated whether SIRT1 inhibitor EX-527 could reverse the anti-nociceptive effect of NAD or resveratrol. It was found that spinal SIRT1 expression, deacetylase activity and NAD/NAM decreased significantly 1, 3, 7, 14 and 21 days after CCI surgery as compared with sham group. In addition, daily intrathecal injection of 5 µl 800 mM NAD 1 h before and 1 day after CCI surgery or single intrathecal injection of 5 µl 90 mM resveratrol 1 h before CCI surgery produced a transient inhibitory effect on thermal hyperalgesia and mechanical allodynia in CCI mice. Finally, an intrathecal injection of 5 µl 1.2 mM EX-527 1 h before NAD or resveratrol administration reversed the anti-nociceptive effect of NAD or resveratrol. These data indicate that the reduction in SIRT1 deacetylase activity may be a factor contributing to the development of neuropathic pain in CCI mice. Our findings suggest that the enhancement of spinal NAD/NAM and/or SIRT1 activity may be a potentially promising strategy for the prevention or treatment of neuropathic pain.

Background Cyclin-dependent kinase 5 (Cdk5) has been shown to play an important role in mediating inflammation-induced heat hyperalgesia. However, the underlying mechanism remains unclear. The aim of this study was to determine whether roscovitine, an inhibitor of Cdk5, could reverse the heat hyperalgesia induced by peripheral injection of complete Freund's adjuvant (CFA) via the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling pathway in the dorsal horn of the spinal cord in rats. Results Heat hyperalgesia induced by peripheral injection of CFA was significantly reversed by roscovitine, TrkB-IgG, and the TrkB inhibitor K252a, respectively. Furthermore, BDNF was significantly increased from 0.5 h to 24 h after CFA injection in the spinal cord dorsal horn. Intrathecal adminstration of the Cdk5 inhibitor roscovitine had no obvious effects on BDNF levels. Increased TrkB protein level was significantly reversed by roscovitine between 0.5 h and 6 h after CFA injection. Cdk5 and TrkB co-immunoprecipitation results suggested Cdk5 mediates the heat hyperalgesia induced by CFA injection by binding with TrkB, and the binding between Cdk5 and TrkB was markedly blocked by intrathecal adminstration of roscovitine. Conclusion Our data suggested that the BDNF-TrkB signaling pathway was involved in CFA-induced heat hyperalgesia mediated by Cdk5. Roscovitine reversed the heat hyperalgesia induced by peripheral injection of CFA by blocking BDNF/TrkB signaling pathway, suggesting that severing the close crosstalk between Cdk5 and the BDNF/TrkB signaling cascade may present a potential target for anti-inflammatory pain.

Postoperative cognitive dysfunction (POCD) is a clinical phenomenon characterized by cognitive deficits in patients after anesthesia and surgery, especially in geriatric surgical patients. Although it has been documented that isoflurane exposure impaired cognitive function in several aged animal models, there are few clinical interventions and treatments available to prevent this disorder. Minocycline has been well established to exert neuroprotective effects in various experimental animal models and neurodegenerative diseases. Therefore, we hypothesized that pretreatment with minocycline attenuates isoflurane-induced cognitive decline in aged rats. In the present study, twenty-month-old rats were administered minocycline or an equal volume of saline by intraperitoneal injection 12 h before exposure to isoflurane. Then the rats were exposed to 1.3% isoflurane for 4 h. Two weeks later, spatial learning and memory of the rats were examined using the Morris Water Maze. We found that pretreatment with minocycline mitigated isoflurane-induced cognitive deficits and suppressed the isoflurane-induced excessive release of IL-1β and caspase-3 in the hippocampal CA1 region at 4 h after isoflurane exposure, as well as the number of TUNEL-positive nuclei. In addition, minocycline treatment also prevented the changes of synaptic ultrastructure in the hippocampal CA1 region induced by isoflurane. In conclusion, pretreatment with minocycline attenuated isoflurane-induced cognitive impairment in aged rats.

Introduction Angiogenesis regulates myometrial spiral artery expansion and remodeling that are key mechanisms for upregulating uterine blood flow during pregnancy. Hydrogen sulfide (H 2 S), mainly synthesized from l -cysteine by cystathionine-beta synthase (CBS) and cystathionine-gamma lyase (CSE), is a potent vasodilator and proangiogenic factor. However, it is unknown if pregnancy regulates H 2 S biosynthesis and if H 2 S plays a role in myometrial angiogenesis. Hypothesis Correlating with endogenous estrogens during the menstrual cycle and pregnancy, H 2 S biosynthesis is augmented to stimulate myometrial angiogenesis in vivo and in vitro. Methods Myometrial tissues were obtained from hysterectomies from women with different endogenous estrogens ( n = 5–7/group), including late pregnant (very high estrogen), premenopausal proliferative (high estrogen) and secretory (low estrogen) of the menstrual cycle, and postmenopausal (very low estrogen). Tissue levels of CBS and CSE mRNA and protein were determined by real-time qPCR and immunoblotting, respectively. Tissue sections were analyzed by immunofluorescence microscopy with antibodies of CBS or CSE with an endothelial marker protein CD31. Images were analyzed for quantifying cellular CBS and CSE protein expression and angiogenesis index. Primary myocytes and microvascular endothelial cells (hMMEC) were enzymatically isolated for in vitro angiogenesis assays. Results Myometrial H 2 S levels and angiogenesis index were greatest in association with significant upregulation of CBS but not CSE mRNA and protein and elevated estrogens in pregnant vs. non-pregnant women. CBS protein was localized in myocytes and microvessels. A slow releasing H 2 S donor significantly stimulated hMMEC proliferation, migration and tube formation in vitro (in vivo angiogenesis). Co-culture with myocytes also stimulated hMMEC migration in vitro, which was blocked by a specific inhibitor of CBS. Conclusion Pregnancy augmented H 2 S biosynthesis due to enhanced CBS expression in myocytes and microvessels stimulates myometrial angiogenesis in the presence of elevated estrogens during the menstrual cycle and pregnancy (Funded by NIH RO1 HL70562 and R21 HL98746).

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.

Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10–30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two-dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, nine were novel SNO-proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation. J. Cell. Physiol. 227: 146–159, 2012. © 2011 Wiley Periodicals, Inc.

Because human duodenal mucosal bicarbonate secretion (DMBS) protects duodenum against acid-peptic injury, we hypothesize that estrogen stimulates DMBS, thereby attributing to the clinically observed lower incidence of duodenal ulcer in premenopausal women than the age-matched men. We found that basal and acid-stimulated DMBS responses were 1.5 and 2.4-fold higher in female than male mice in vivo, respectively. Acid-stimulated DMBS in both genders was abolished by ICI 182,780 and tamoxifen. Estradiol-17beta (E2) and the selective estrogen receptor (ER) agonists of ERalpha [1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole] and ERbeta [2,3-bis(4-hydroxyphenyl) propionitrile], but not progesterone, rapidly stimulated ER-dependent murine DMBS in vivo. E2 dose dependently stimulated murine DMBS, which was attenuated by a Cl(-)/HCO3(-) anion exchanger inhibitor 4,4'-didsothio- cyanostilbene-2, 2'-disulfonic acid, removal of extracellular Cl(-), and in cystic fibrosis transmembrane conductance regulator knockout female mice. E2 stimulated murine DMBS in vitro in both genders with significantly greater response in female than male mice (female to male ratio = 4.3). ERalpha and ERbeta mRNAs and proteins were detected in murine duodenal epithelium of both genders; however, neither ERalpha nor ERbeta mRNA and protein expression levels differed according to gender. E2 rapidly mobilized intracellular calcium in a duodenal epithelial SCBN cell line that expresses ERalpha and ERbeta, whereas BAPTA-AM abolished E2-stimulated murine DMBS. Thus, our data show that E2 stimulates DMBS via ER dependent mechanisms linked to intracellular calcium, cystic fibrosis transmembrane conductance regulator, and Cl(-)/HCO3(-) anion exchanger. Gender-associated differences in basal, acid- and E2-stimulated DMBS may have offered a reasonable explanation for the clinically observed lower incidence of duodenal ulcer in premenopausal women than age-matched men.

Finland blue fox (Alopex lagopus) has great reputation in pelt industry around the world for its large size and top-ranking fur quality; however, both the herd size and the average survival rate of purebred offspring are rather low in production systems in China. Surgical transfer of blue fox embryos was investigated as a means to increase the population fox and also as a possible means to conserve endangered canine species. The animals were chosen on the basis of synchrony in natural oestrus. During the reproductive season of blue fox, 59 embryos were flushed from 6 farmed donors 9-11 days after the first insemination, and 53 embryos were transferred surgically into the uteri of the 6 paired recipients with natural synchronized oestrous. Two of the recipients littered 46-49 days after embryo transfer; one gave birth to 7 pups and the other 1 pup. This report describes the first successful embryo transfer in the farmed blue fox in China.

Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. c-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P

Cyclin-dependent kinase 5 is a proline-directed serine/threonine kinase and its activity participates in the regulation of nociceptive signaling. Like binding with the activators (P35 or P25), the phosphorylation of Cdk5 plays a critical role in Cdk5 activation. However, it is still unclear whether Cdk5 phosphorylation (p-Cdk5) contributes to pain hyperalgesia. The aim of our current study was to identify the roles of p-Cdk5 and its upstream regulator in response to peripheral inflammation. Complete Freund's adjuvant (CFA) injection induced acute peripheral inflammation and heat hyperalgesia, which was accompanied by sustained increases in phospho-ERK1/2 (p-ERK1/2) and phospho-Cdk5(S159) (p-Cdk5(S159)) in the spinal cord dorsal horn (SCDH). CFA-induced p-ERK primarily colocalized with p-Cdk5(S159) in superficial dorsal horn neurons. Levels in p-ERK and p-Cdk5 were also increased in the 2(nd) phase of hyperalgesia induced by formalin injection, which can produce acute and tonic inflammatory pain. MAP kinase kinase inhibitor U0126 intrathecal delivery significantly suppressed the elevation of p-Cdk5(S159), Cdk5 activity and pain response behavior (Heat hyperalgesia, Spontaneous flinches) induced by CFA or formalin injection. Cdk5 inhibitor roscovitine intrathecal administration also suppressed CFA-induced heat hyperalgesia and Cdk5 phosphorylation, but did not attenuate ERK activation. All these findings suggested that p-Cdk5(S159) regulated by ERK pathway activity may be a critical mechanism involved in the activation of Cdk5 in nociceptive spinal neurons contributes to peripheral inflammatory pain hypersensitivity.

Mechanisms associated with cyclin-dependent kinase 5 (Cdk5)-mediated heat hyperalgesia induced by inflammation remain undefined. This study was designed to examine whether Cdk5 mediates heat hyperalgesia resulting from peripheral injection of complete Freund's adjuvant (CFA) in the spinal dorsal horns of rats by interacting with synaptophysin, a well known membrane protein mediating the endocytosis-exocytosis cycle of synaptic vesicles as a molecular marker associated with presynaptic vesicle membranes. The role of Cdk5 in mediating synaptophysin was examined through the combined use of behavioral approaches, imaging studies, and immunoprecipitation following CFA-induced inflammatory pain. Results showed that Cdk5 colocalized with both synaptophysin and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) consisting of VAMP-2, SNAP-25, and syntaxin 1A in spinal dorsal horn of rats. Increased synaptophysin expression of spinal cord horn neurons post intraplantar injection of CFA coincided with increased duration of heat hyperalgesia lasting from 6 h to 3 d. Intrathecal administration of roscovitine, a Cdk5 specific inhibitor, significantly depressed synaptophysin expression during peak heat hyperalgesia and heat hyperalgesia induced by peripheral injection of CFA. Data presented in this report indicated that calpain activity was transiently upregulated 6 h post CFA-treatment despite previous reports suggesting that calpain was capable of cleaving p35 into p25. Results from previous studies obtained by other laboratories demonstrated that significant changes in p35 expression levels within spinal cord horn neurons were not observed in the CFA-treated inflammatory pain model although significant upregulation of Cdk5 kinase was observed between 2 h to 7 d. Therefore, generation of p25 occurred in a calpain-independent fashion in a CFA-treated inflammatory pain model. Our results demonstrated that increased synaptophysin levels were involved in heat hyperalgesia mediated by Cdk5 in spinal cord dorsal horns of CFA-treated rats, suggesting that inhibiting abnormal activation of Cdk5-synaptophysin may present a novel target for diminishing inflammatory pain.