Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypo-glycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner car development and growth in zebrafish.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotem nm23, crustacyanin-Cl and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (K-d) of 880 nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400 nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott-Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. (C) 2010 Elsevier Ltd. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Typhoon effects on DOC dynamics in a phosphate-limited reservoir

To explore typhoon effects on dissolved organic carbon (DOC) dynamics, field investigations (tributary and dam site) and laboratory experiments (bioassay and DOC consumption) were conducted in a subtropical reservoir. A tributary survey indicated that after typhoon disruption, upstream areas were the sources of phosphate (P) but not DOC for the dam site located downstream. Bioassay experiments verified P-limitation on bacteria and phytoplankton during summer stratification, and bacteria showed a faster response than algae to added P. Experiments indicated that DOC consumption was determined by the availability of P. The 4 yr typhoon period (June-September) data of the dam site denoted that DOC concentration (27 to 270 mu M C) and its rate of change (-13 to 24 mu M C d(-1)) varied more dramatically in the weak (2006 and 2007) than in the strong (2004 and 2005) typhoon years. The negative correlation of DOC with the ratio of bacterial production (BP) to primary production (PP) in the euphotic zone (0 to 10 m) signified the interactive effects of auto- and heterotrophic processes on DOC variation. In the aphotic zone, the variation of DOC could be ascribed to the change of BP, which showed a positive correlation with P concentrations. This study documents that DOC concentration in the studied system varied at multiple time scales. Such variation can be explained by the decoupling between BP and PP, which is believed to be a function of the limiting nutrient's availability. More importantly, this study suggests that the P supply introduced by strong typhoons might have substantiated a tighter coupling between BP and PP, so that the amplitude of DOC oscillation during the summer period was effectively reduced.

Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration- and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 mu M and 50 mu M for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

磁层-电离层耦合现象的分析研究

The magnetosphere-ionosphere coupling is mainly manifested by the trans- porting processes of energy into the ionosphere , the energy is carried by solar wind and firstly accumulate at the magnetosphere, and the coupling processes also significantly include the interaction between the magnetosphere and ionosphere for mass and energy. At the quiet condition, energy is delivered by the large-scale convection of the geomagnetic field; the huge energy from solar wind bulk will be injected into and consumed at the near magnetosphere and ionosphere by the geomagnetic storm and substorm activities. Aurorae and FACs (Field-aligned currents) are the important phenomena in the coupling processes. In the present work, firstly, we analyze the activity characteristics of auroral precipitating particle, secondly, we study the distribution characters of large-scale field aligned currents (LS FACs) at storm-time using the observations from different satellites at different altitudes. Finally, we investigate the evolution of the geomagnetic field configuration at the nightside sector on the onset of the expansion phase in a substorm event, the substorm event happened at 0430UT to 0630UT on 8th Nov. 2004. The main results as follows： At the first, the data of the estimated power input (EPI) of auroral particles from NOAA/POES (Polar orbiting environmental satellite) for some 30 years have been analyzed. The variation tendencies of the EPI generally coincide with aa, AE and Dst indices. The annual variation of EPI shows equinox peaks and an asymmetric-activity with a higher peak in the winter-hemisphere than in the summer-hemisphere. The diurnal UT variations are different from north and south hemisphere: for north hemisphere, the peak appears at 1200UT, and the relative deviation is 22% to the daily average of the north hemisphere. For south hemisphere, the maximal deviation is 22% at 2000UT. So the diurnal variation of EPI is more dominant than the annual variation which maximal deviation is 3% to 12% for different seasons. Studies on correlations of the hourly average of EPI, Pa, with AE and Dst indices show a correlation coefficient r=0.74 of Pa and AE, and r=-0.55 of Pa and Dst. The hourly EPIs for north and south polar regions, NPa and SPa, show a north-south asymmetry with a higher correlation of SPa and AE (or Dst). Time delays of EPI with respect to magnetic indices are examined, the maximum correlation coefficient of Pa with AE (r=0.78) occurs when the time delay =0, suggesting a synchronous activity of auroral electrojet and auroral precipitating particles, while =1-2h, the correlation coefficient of Pa with Dst is maximum (r=0.57), suggesting that the activity of auroral particle precipitating may influence the ring current on some extent. Sencondly, we use the high-resolution magnetic field vector data of the CHAMP satellite to investigate the distribution of large-scale FACs during the great magnetic storm on 7th to 8th Nov. 2004. The results show that, whether in the northern or southern hemisphere, the number and density of large-scale FACs during the main-phase are more and bigger than these during the recover-phase, and the number of large-scale FACs in morning sector obviously is more than that in afternoon sector. In terms of the magnetic indices, we find that large-scale FACs in morning sector significantly affected by the substorm activities, while in afternoon sector the large-scale FACs mainly indicate the fluctuations of the ring-current in storm time. Accordingly to the former studies, similarly, we find that in the morning sector, the scale of the large-scale FACs move to the high-latitude region, and in the afternoon sector, large-scale FACs distinctly expand to the low-latitude region. During the time periods that the NOAA/POES auroral precipitating particle power data temporally correspond to the large-scale FACs, the more the power of auroral particle is, the more and bigger the number and density of FACs are. At the same time, we use the magnetic field vector data of POLAR obtain a good form of region 1, region 2, and three pieces of cusp FACs during a single transit at 1930UT-2006UT on 07th. And the characteristics of simultaneous electric field and energy particles observations on Polar are coincide with the five FACs pieces. Finally, by means of the observation of Cluster 4 and Goes 10、 Goes 12, we analyze the evolution process of the change of the magnetic field configuration at night sector at the expansion phase of a substorm event which happened during 0430UT to 0630UT on 8th Nov. 2004, we find that the times of the beginning of the polarizations of magnetic field are observed from Goes 10 to Goes 12 then to Cluster 4. So, at the synchronous orbit ( 6.6 RE) to 10RE distance scale of the neutral sheet, the current disruption spread tailward. Simultaneously, the strengthen of the FACs deduced from these satellites’ magnetic field observations are almost consistent with the times of polarizations, as well as the high energy particles injection and the electric field dominant variation. The onset times determined by the magnetic field polarizations from these satellites are all ahead of the onset time that confirmed from the auroral electrojet indices. So, these characters of different observations can be used as the criterions to determine the onset time for the substorms of such type as we studied.

细胞外信号调节蛋白激酶-cAMP反应元件结合蛋白信号通路在应激所致抑郁行为障碍中的作用

Stress is the most important factor in the vulnerability to depression and other behavioral disorders, but the mechanisms that stress signals are transferred into depression are far from understanding. To date, the neurotransmitters, neurotrophins and signal pathway have been concerned in the topic focusing on the pathophysiology of depression, but there are still many puzzles. Increasing evidence has indicated that the alteration in neuronal plasticity is the “trace” of stress-induced damages. The extracellular signal-regulated protein kinase(ERK)-cyclic-AMP-responsive element（CRE）-binding protein(CREB)signal pathway is a powerful intracellular signal transduction pathway participating in neuronal plasticity which is involved in higher brain cognitive functions such as learning and memory. However, so far, little is known about the role of the ERK-CREB signal pathway in response to stress and emotional modulations. Thus the aim of the study was to systematically investigate the role of the ERK-CEB signal pathway in depressive-like behaviors induced by stress. Depression animal models, antidepressant agent treatment and disruption of signal pathway in specific brain regions were applied. In the present study, three experiment sessions were designed to make sure whether the ERK-CREB signal pathway was indeed one of pathophysiological mechanisms of depressive-like behaviors induced by stress. In experiment one, two different stress animal models were applied, chronic forced swim stress and chronic empty water bottle stress. After stress, all animals were tested behaviorally using open-field, elevated-plus maze and saccharine preference test, and brain samples were processed for determination of ERK, P-ERK, CREB and P-CREB using western blot. The relationships between the proteins of ERK, P-ERK, CREB and P-CREB in the brain and the behavioral variables were also analyzed. In experiment two, rats were treated with antidepressant agent fluoxetine once a day for 21 consecutive days, then the brain levels of ERK, P-ERK, CREB and P-CREB was determined, the depressive-like behaviors were also examined. In experiment three, mitogen activated extracellular-signal-regulated kinase kinase (MEK) inhibitor U0126 was administrated to inhabit the activation of ERK in the hippocampus and prefrontal cortex respectively, then behavioral measurements and protein detection were conducted. The main results of the study were as the following: (1) Chronic forced swim stress induced animals to suffer depression and disrupted the ERK-CREB signal pathway in hippocampus and prefrontal cortex. There were significant correlations between P-ERK2, P-CREB and multiple variables of depressive-like behaviors. (2) Chronic empty water bottle stress did not induce depressive-like behaviors. Such stress decreased the brain level of P-ERK2 in hippocampus and prefrontal cortex, but the level of P-CREB in the hippocampus was increased. (3) The antidepressant agent fluoxetine relieved depressive-like behaviors and increased the activities of the ERK-CREB signal pathway in stressed animals. (4) Animals treated with U0126 injection into hippocampus showed decreased activities of the ERK-CREB signal pathway in the hippocampus, and suffered depression comorbid with anxiety. (5) Animals treated with U0126 injection into prefrontal cortex showed decreased activities of the ERK-CREB signal pathway in the prefrontal cortex, and exhibited depressive-like behaviors. In conclusion, The ERK-CREB signal pathway in the hippocampus and prefrontal cortex was involved in stress responses and significantly correlated with depressive-like behaviors; The ERK-CREB signal pathway in the hippocampus and prefrontal cortex participated in the mechanism that fluoxetine reversed stress-induced behavioral disorders, and might be the target pathway of the therapeutic action of antidepressants; The disruption of the ERK-CREB signal pathway in the hippocampus or prefrontal cortex led to depressive-like behaviors in animals, suggesting that disruption of ERK-CREB pathway in the hippocampus or prefrontal cortex was involved in the pathophysiology of depression, and might be at least one of the mechanisms of depression induced by stress.