Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

Role of αVβ3 integrin in embryo implantation in the mouse

Integrin, a heterodimeric adhesive molecule composed of α and β subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the “implantation window” stage, α Vβ 3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that α Vβ 3 integrin was clearly expressed in the mouse blastocyst. Injection of α Vβ 3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, α Vβ 3 integrin antisera at 1︰100 and 1︰200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of α Vβ 3 integrin antiserum was dosage/dilution-dependent. Thus, α Vβ 3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at “implantation” stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.     

Role of Ppt1 in multiple stress responses in Candida albicans

To study the function of CaPptl, we deleted PPT1 gene from the Candida albicans genome by sequentially replacing the entire coding region with the selectable markers ARG4 and HIS1. The results showed that the deletion of Pptl did not affect the hyphal formation of C. albicans under serum induction and caused enhanced sensitivity to DNA damage, Calcofluor white and salt- induced stress. We also found that Pptl was not required for the phenotypic response of cells treated with the genotoxins, methylmethane sulfonate and hydroxyurea. Flow cytometric analyses indicated that pptlA cells and wild-type cells showed similar G2/M arrest profiles when exposed to DNA damage stress. Pptl was not required for the activation of the DNA damage response pathway, as indicated by normal phosphorylation of Rad53 and Rfa2 in pptlA cells under DNA damage stress. We suggest that Pptl plays important roles in response to various stress conditions in C. albicans.     

Study on the DME-cytochrome<Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> and its mutants at site of F58

Phenylalanine-58 is one of the conservative residues in the hydrophobic pocket of Cyt bs, which forms aromatic stacking with the heme b. Previous study showed that both the stacking and the property of the aromatic residue affect hydrophobicity of the heme pocket, leading to change of protein‘s property. In order to further reveal the essence we esterify the heme propionate of Cyt bs, F58Y and F58W, and eliminate the hydrogen bond between heme propionate and Ser64 in examining the effect of hydrogen net on the π-π interaction. In this paper thermal denaturation of DME-Cyt b5 and its F58Y and F58W mutants has been studied by UV-visible and CD spectra. The heme transfer reactions between these proteins and apo-myoglobin have been studied as well. The results demonstrate that esterification did not destroy the aromatic stacking; however, it affects the stability of the proteins due to different volumes, hydropho-bicities and hydrogen bonds forming ability of these substituents.     

Isolation and characterization of 4 gametophytic male sterile mutants in Arabidopsis thaliana

In flowering plants, male fertility depends on the formation and development of normal male gametophytes or pollen grains. However, little is known about the molecular mechanisms that regulate the processes. Here, we report the identification of four novel independent Arabidopsis gametophytic male sterile mutants, apam1, apam2, apam3 and apam4 (Arabidopsis pollen abortion mutant). The four mutants that were generated by the insertions of geneand enhancer-trap Ds transposon elements were defective in pollen development. Genetic analysis results showed that all four mutations resulted in the loss of male gametophytic function, but did not affect female gametophytic function, and the Ds elements were linked to the mutations tightly in all four mutants. Localization of the Ds insertion sites by thermal asymmetric interlaced PCR (TAIL-PCR) showed that the Ds elements were inserted in four different loci distributed on three chromosomes, chromosomes II, III and V. In summary, the apam4 is allelic to AHA3, while the other three were located in places where there are no genes that have been known to be involved in pollen development, suggesting that they are novel mutations involved in pollen development.     

Effects of anthropogenic disturbance on richness-dependent stability in Napahai plateau wetland

Unique plateau wetlands in China provide essential ecosystem functions and services and influence the health,environment and security of the downstream regions.In recent years,these plateau wetlands have experienced significant anthropogenic disturbance,but studies that evaluate the effects of such disturbance on ecological stability are rare.Our study tested how three typical types of human-related activities affect plant richness and ecological stability in Napahai plateau wetland,Shangri-La,China.The results showed that the anthropogenic disturbance had a direct effect on richness,and an indirect effect on stability mediated by richness.Anthropogenic disturbance did not alter the positive relationship between plant richness and community stability,and the stabilizing effect of richness could be explained by statistical averaging,overyielding effect,and component population stability.Our study complements previous studies that tested the richness-stability relationships in synthesized assemblages with richness specifically manipulated and studies that introduced mowing treatment to mimic real anthropogenic disturbance.The results further suggest that necessary steps,such as anthropogenic disturbance mitigation and plant richness conservation,are urgently required for maintaining healthy plateau wetlands and for sustaining their ecosystem functions and services.     

Interaction between PⅡ and NifA in Azospirillum brasilense Sp7

The interaction between PⅡ and NifA in A.brasilense Sp7 was investigated by using the yeast two-hybrid system.Our experimental results showed that PⅡ directly interacted with the entire NifA protein and its N-terminal domain,but did not interact with the central domin and the C-terminal domain of NifA.No interaction happened if glnB coding for PⅡ was frame-shift mutated.Pz,a homolog of PⅡ,had no interation with NifA.     

Expression of LIF,VEGF, CD57 and CD68 after the transfer of rat embryos to mouse uteri

The high failure rate of interspecific preganacy is a major obstacle to the successful interspectific cloning of mammals,To in vestigate the reasons for the failure of inter-specfic pregnancy between rats and mice,we transferred rat blastocysts into mouse uteri on the third day of pseudopreg -nancy (D3),oure previous study showed that intact rat embryos could still be observed in mouse uteri on D9.In the present study ,we found that expression of CD57 and CD68 increased significantly at the maternal -fetal interface fol-lowing the transfer of rat embryos,Similarly ,Leukaemia inhibitory factor(LIF) expression increased ,but vascular endothelial growth factor(VEGF) expession decreased,In a co-culture system ,the percentage of rat ectoplacental cones (EPCs) with adhesion and outgrowth and outgrowth area on mouse uterine decidual cells were less than that of mouse EPCs,These results indicate that an increase in the immunological rejection response and a decrease in the in vasiveness of rat embryos may be important reasons for the failure of interspecific pregnancy between rat and mouse.     

Comparison of the Photosynthetic Characteristics of Two Developmental Stages in Nostoc sphaeroides Kutzing （Cyanophyta）

The photosynthetic activities between two main developmental stages, colony and hormogonium, of the edible cyanobacterium Nostoc sphaeroides Kutzing, were compared. Hormogonia have a higher content of chlorophyll than that of colonies. It showed that the ratios of phycocyain （PC）, allophycocyain （APC） and phycoerythrocyanin （PEC） in hormogonia and colonies were different. The room temperature chlorophyll fluorescence, 77 K chlorophyll fluorescence, measurements of PSⅠand PS Ⅱ activities all showed that colony has higher photosynthetic competence than hormogonia. Hormogonia had a higher respiration rate than colony, while their maximum photosynthetic oxygen evolution rates were very close. The responses of hormogonia and colonies to high light illuminations also were different. Both of their oxygen evolution rates decreased quickly with the prolonged high light illumination, but hormogonia can keep relatively higher PSⅡ activity （Fv/Fm） than that of colonies. The results suggested that colony was photosynthetically more competent than hormogonia, while the ability of hormogonia to tolerate high light illumination was higher than that of colony.     

Aberrant DNA methylation patterns in cultured mouse embryos

Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.     

Induction of matrix metalloproteinase-9 and -2 activity in mouse blastocyst by fibronectin-integrin interaction

Fibronectin, a major extracellular matrix, plays an important role in embryo implantation by mediating embryo adhesion and outgrowth. In this work, mouse blastocysts produced pro-matrix metalloproteinase-9, pro-matrix metalloproteinase-2 and 64 ku matrix metalloproteinase-2 when they were co-cultured with fibronectin. In contrast, mouse blastocysts did not produce these proteinases without fibronectin. Focal adhesion kinase is a fundamental molecule of integrin signaling pathway and its antisense oligodeoxynucleiotide inhibited blastocyst matrix metalloproteinases expression induced by fibronectin. The results indicated that fibronectin triggered matrix metalloproteinase-9 and -2 expression in mouse blastocyst through its integrin receptors and subsequent signaling pathway, which enhanced the synchronization of blastocyst invasiveness and uterine receptivity and ensured the accuracy of events relative to implantation in timing and spatiality.     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

中药止血散的止血作用初探

本实验选用ＮＩＨ小鼠，随机分成Ⅰ、Ⅱ、Ⅲ三组，用环磷酰胺建立小白鼠再生障碍性贫血动物模型．实验结果显示：出血时间Ⅰ（对照）组（１．５８±０．１５）ｍｉｎ，Ⅱ（环磷酰胺）组（５．２３±０．２７）ｍｉｎ，Ⅲ（环磷酰胺＋止血散）组（１．５７±０．１）ｍｉｎ；统计学处理表明Ⅱ与Ⅰ组间有差异（ｐ＜０．０５），Ⅱ与Ⅲ组间有差异（ｐ＜０．０５），Ⅰ与Ⅲ组间无差异（ｐ＞０．０５）；血块回缩分别为完全、不良、完全，血小板计数分别为４３０．２±３４．３６×１０￣９Ｌ￣（－１），２８４±１９．９６×１０￣９Ｌ￣（－１），４６５．４±２２．４８×１０￣９Ｌ￣（－１）．统计处理表明Ⅱ与Ⅰ组间和Ⅱ与Ⅲ组间存在显著性差异（ｐ＜０．０１），Ⅰ与Ⅲ组间无差异，本实验提示：中药制剂止血散能促进血小板的生成和血小板的释放，具有止血作用．     

DanHong Injection inhibits the development of primary abdominal aortic aneurysms in apoE knockout mice

Clinical observations indicate that DanHong Injection （DHI） can increase blood flow and reduce various syndromes in patients with cardiovascular disease. How- ever, it still needs to define the function of DHI and the involved mechanisms in details, such as the protective effect on the development of primary abdominal aortic aneurysms （AAAs）. In this study, we determined whether DHI is able to inhibit AAA in apoE knockout （apoE-/-） mice. Thirty apoE-/- male mice on high-fat diet （0.5 % cholesterol, 21% fat） were randomly divided into two groups and received i.p. injection of saline （100 μL/day） and DHI （100 μL/day）, respectively, for 16 weeks. At the end of experiment, we determined the development of atherosclerosis in en face aorta and aneurysms,pathological morphology of arterial wall, and serum lipid levels. We also determined the expression of monocyte chemoattractant protein-1 （MCP-1）, MMP-2, and MMP-9 mRNA in aortic wall using real-time RT-PCR. Our results indicated that high-fat diet induced the development of AAAs in apoE-/- mice, but the induction was totally blocked by DHI （P 〈 0.01）. The result of staining of abdominal aortic cross sections showed that DHI main- tained the collagen content in arterial wall, thereby pre- venting the animals from the development of AAA. Although DHI had little effect on serum total- and LDL- cholesterol levels, it reduced the expression of MCP-1, MMP-2, and MMP-9 mRNA in aortic wall （P 〈 0.01）. Taken together, our study suggests that DHI can inhibit the high-fat diet-induced AAA formation. The inhibitory effects may be related to the maintenance of the collagen content and inhibition of expression of AAA-related genes. Our study may suggest a new application of DHI in clinics.     

Effect of matrix metallo-proteinase-26 (MMP-26) during embryo implantation in the mouse

Matrix metalloproteinase-26 (MMP-26, endometase and matrilysin-2), a novel member of the MMPs family, is detected not only in the placenta and uterus, but is widely expressed in malignant tumors from different sources as well as in diverse tumor cell lines. However, the function of MMP-26 in the reproductive system has never been reported. Expression of MMP-26 in mouse embryos and the function of the MMP-26 antibody during mouse embryo implantation was examined for the first time by injecting the uterine horn, immunohistochemistry,in situ hybridization, co-culture of mouse blastocysts and uterine monolayer epithelial cells, Western blot, RT-PCR, Northern blot and zymography. Our results show that there is strong expression of MMP-26 mRNA and protein in the mouse embryo. Furthermore, the MMP-26 antibody dramatically inhibited mouse embryo implantation and significantly inhibited adhesion and outgrowth of mouse blastocysts onin vitro uterine monolayer epithelial cells. At the same time, the MMP-26 antibody inhibited the expression of integrin αV mRNA and protein in a dose-dependent manner. These data suggest that MMP-26 may play a role in some of the tissue-remodeling events associated with the invasion of the endometrium by trophoblast cells and facilitate successfully embryo implantation.     

Regulation of embryo implantation by nitric oxide in mouse

Intrauterine injection and zymography were used to investigate the effect of nitric oxide (NO) on embryo implantation in mice. On day 3, one uterine horn of female pregnant mice was injected intraluminally with various doses of nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), while the contralateral horn served as control. Animals were sacrificed by cervical dislocation on day 7 of gestation, and the number of implanted embryos in each horn was calculated. The results showed that lower doses (0.05 mg L-NAME) did not inhibit implantation significantly (P > 0.05), but high doses (0.2 mg L- NAME) resulted in a significant reduction in the number of implanted embryos (P < 0.05). Co-administration of SNP, a generator of NO, with L-NAME would reverse the antiimplantation effect of L-NAME. To further understand the precise mechanism of NO in implantation, matrix metalloproteinase (MMPs) activities were detected by gelatin zymography. The reduction in the number of implanted embryos in 0.2 mg L-NAME treated group was associated with decreased MMP-9 activity but a stable MMP-2 activity. The activities of MMP-2 and MMP-9 were not changed in L-NAME and SNP treated group. These data suggest that NO acts as a mediator to regulate the activity of MMP-9, and facilitates embryo implantation.     

IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.     

人脑胶质瘤中MMP-9的表达及意义

目的：探讨MMP-9在人脑胶质瘤中的表达及意义。方法：采用免疫组化S-P法检测45例人脑胶质瘤及32例瘤旁正常脑组织中MMP-9蛋白的表达。结果：MMP-9在正常脑组织、低度恶性及高度恶性脑胶质瘤组织中的阳性表达率分别为15.6%,45.8%,81.0%。结论：MMP-9蛋白表达与胶质瘤的发生和恶性进展有关。