Production and Epitope Specificky of Monoclonal Antibody against Mouse Peptidylarginine Deiminase Type II

Peptidylarginine deiminase catalyzes the conversion of arginyl residues in proteins to citrullyl residues in the presence of Ca²⁺. We described the preparation of monoclonal antibody (subclass type IgG₁) specific to mouse peptidylarginine deiminase type II. The antibody had no effect on the enzyme activity and its specific epitope was localized in the eight-residue segment at the amino-terminal portion of the enzyme.     

胶原酶-3活性在大鼠动情周期及胚泡着床中的变化(英文)

大鼠动情周期以及胚胎着床过程中,子宫内膜会发生结缔组织的降解与重构。胶原酶3（MMP－13）是降解纤维类胶原的主要蛋白水解酶类之一。其活性在这些过程中的变化值得研究。采用液体闪烁计数测定~3H标记胶原的方法,对大鼠动情周期及早期妊娠子宫中胶原酶-3（MMP－13）的活性进行了测定。结果表明：在动情周期中;激活型MMP－13在间情期最低,酶原型及激活型的MMP－13在动前期达高峰,动情后期酶原型和激活型MMP也明显高于间情期（P＜0.05）（Fig．1）。妊娠第1、2天酶原型的MMP-13的活性显著高于第3～7天,第3、4天酶原型和激活型MMP-13的活性均低于妊娠第1、2天（P＜0.05）;而第5天酶原型MMP-13的活性却显著高于第4、6两天（P＜0．05）;激活型MMP－13的活性也高于第4天（P＜0．05）（Fig．2）。着床部位酶原型MMP－13的活性明显高于非着床部位（P＜0．05）,而激活型MMP－13的活性则无明显差异（P＞0．05）（Fig．3）。大鼠假孕早期子宫中MMP－13的活性变化与正常早期妊娠相似,但其活性却明显低于正常早期妊娠（Fig．4）。结果提示：大鼠子宫中MMP-13参与大鼠动情周期及早期妊娠过程,尤其是在胚胎着床过程中可能起着重要作用。     

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.     

Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brown^Dominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.     

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

This study investigated the functional roles of the N-terminal Ca²⁺ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca²⁺-binding site of PAD4 were mutated to disrupt the binding of Ca²⁺ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k _cat/K _m,BAEE values were 0.02, 0.63 and 0.01 s⁻¹mM⁻¹ (20.8 s⁻¹mM⁻¹ for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k _cat value of 0.3 s⁻¹ (13.3 s⁻¹ for wild-type), whereas D176A retained some catalytic power with a k _cat of 9.7 s⁻¹. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k _cat/K _m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca²⁺ indicated that the conformational stability of the enzyme is highly dependent on Ca²⁺ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca²⁺ ions in the N-terminal Ca²⁺-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca²⁺ ions play critical roles in the full activation of the PAD4 enzyme.     

基质金属蛋白酶-2、-9及其组织抑制因子(TIMPs)在动情周期大鼠子宫中的表达(英文)

基质金属蛋白酶（ＭＭＰｓ）家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（ＴＩＭＰｓ）的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过ＭＭＰｓ和ＴＩＭＰｓ两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从ＭＭＰｓ和ＴＩＭＰｓ两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将其处死、取子宫制备酶粗提液和组织切片,采用酶谱法（ｚｙｍｏｙｒａｎｈｎ）和原位杂交方法研究动情周期大鼠子宫中ＭＭＰ－２和－９的活性变化以及ＭＭＰ－２、－９和ＴＩＭＰ－１、－２、－３ｍＲＮＡ的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Ｔａｂｌｅ１。酶谱结果显示：在动情周期大鼠子宫中只检测到６７ｋＤａ的ＭＭＰ－２活性,而没有检测到ＭＭＰ－９的活性（Ｆｉｇ．１）。ＭＭＰ－２的活性在动情前期最高,动情期和动情后期次之,间情期最低（Ｆｉｇ．２）。原位杂交结果显示：ＭＭＰ－２、－９、ＴＩＭＰ－１、－２、－３ｍＲＮＡ主要在子宫内膜基底部的基质细胞中表达。ＭＭＰ－２和－９ｍＲＮＡ在动情前期、动情期和动     

Histomorphological Changes in the Reproductive Tract of Female Hemiechinus auritus collaris,Gray in Relation to the Estrous Cycle

The average length of the estrous period is 7.9 days in Hemiechinus auritus collaris. Follicular development takes place cyclically. Maximum atresia of follicles is noticed during metestrus. The corpus luteum is formed by the shrinkage of the remaining granulosa cells of the ovulated follicle. Maximum development of the corpus luteum is seen during the pregnancy. Histological changes in the uterus and vagina during the estrous cycle are described. There is a marked rise in the weight of the uterus and ovary during the proestrus phase of the cycle. The estrus phase is characterized by the signs of degeneration in the uterine epithelium. During metestrus degeneration and regeneration proceed together. Secretory activity is at a minimum and the lumina of the glands are empty during diestrus.     

Performing Vaginal Lavage,Crystal Violet Staining,and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.     

Differential Expression and Regulation of Basigin Gene Mouse Uterus during Early Pregnancy

Differential Expression and Regulation of Basigin Gene Mouse Uterus during Early Pregnancy     

Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.     

亚硒酸钠对西门塔尔母牛发情周期生殖激素分泌的影响

选用12头18月龄,体况良好,体重380 kg的西门塔尔牛育成母牛,采用完全随机区组设计分为4组,研究亚硒酸钠(0、0.3、0.6和0.9 mg Se/kg DM)对发情周期外周血清促黄体素、促卵泡素、孕酮和雌二醇分泌的影响。结果表明:日粮添加亚硒酸钠后发情周期促黄体素、促卵泡素、孕酮和雌二醇分泌水平提高,0.3 mg/kg组和0.6 mg/kg组显著高于对照组(P<0.05),0.3 mg/kg组较0.6 mg/kg组高(P>0.05)。根据试验结果推断以亚硒酸钠为硒源,添加0.3 mg Se/kg DM对发情周期生殖激素分泌有显著促进作用,兼顾基础日粮的含硒量,建议日粮硒水平为0.37 mg Se/kg DM。     

Role of Luteinizing Hormone in Regulation of the Rat Estrous Cycle

The role of luteinizing hormone (LH) in regulation of secretionof ovarian steroids during the preovnlatory phase of the ratestrous cycle has been discussed. Evidence indicating an involvementof these steroids in control of the uterine growth, vaginalcornification, mating behavior and LH secretion which occurduring this phase has been reviewed. A positive feedback controlsystem has been proposed in which both ovarian and adrenal steroidsparticipate to initiate the preovulatory "LH surge" which culminatesin ovulation and luteinization     

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26°C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42°C. At 34 and 38°C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42°C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38°C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38°C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.     

Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD)

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.     

Non-identical Distribution Pattern of Epidermal Growth Factor and Platelet-derived Growth Factor in the Mouse Uterus During the Oestrous Cycle and Early Pregnancy

In the present study, we examined by immunohistochemistry the cell-specific distribution of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in the mouse uterus during the oestrous cycle and throughout the first 7 days of pregnancy. Paraffin-embedded tissue samples were immunostained using the avidin–biotin peroxidase technique and then examined by light microscopy. Our results showed that immunostaining for EGF was detected in the stroma but not in the luminal or glandular epithelium. A high concentration of EGF was detected in the stroma around the time of embryo implantation at days 3, 4 and 5 of pregnancy. The implanted embryo at day 7 of gestation showed immunostaining for EGF between the ectoderm and endoderm layers. The cell distribution pattern for PDGF was found to be different from that observed with EGF. Luminal and glandular epithelia displayed PDGF immunostaining throughout the first 7 days of pregnancy, with the highest intensity at days 4 and 5 of gestation. In contrast, no immunostaining was observed in the luminal and glandular epithelia at post-oestrus, dioestrus and pro-oestrus stages. However, a weak reaction started to appear at oestrus. The embryo at the blastocyst stage displayed a strong immunoreaction for antibody against PDGF. In addition, the decidual boundary zone surrounding the implanted embryo at days 5, 6 and 7 of gestation also showed an immunostaining for PDGF. The present observations demonstrate clearly the presence of EGF and PDGF in the mouse uterus in high concentrations at the peri- implantation period. Thus, our results, together with what is known about the effect of EGF and PDGF in controlling the growth, differentiation and activation of a variety of cell types, suggest a possible role for these growth factors during the preparation of the endometrium for implantation in controlling the proliferation activity of stromal and/or epithelial cells.     

Social Control of the Ovarian Cycle and the Function of Estrous Synchrony

SYNOPSIS. The social signals among groups of females can eitherenhance or suppress ovariancyclicity. The ovarian cycle is notunitary, but is instead the integrated product of several differentcomponents which are each affected by social signals of differentmodalities. This interaction between female behavior and ovariancycle components has different manifestations in different species.Depending on its temporal context and the social and physicalenvironment, the same behavior/hormone interaction can takedifferent forms. In some contexts, these interactions can beadaptive for the individual. In others, they can generate astrong epiphenomenon or artifact that may not confer a directadaptive advantage itself, but still be necessary for otheraspects of the coordination between social behavior and reproduction.     

Changes in Ribosome-associated Proteins during Sea Urchin Development

Ribosomes isolated from unfertilised eggs of the sea urchin, Strongylocentrotus purpuratus , have a higher protein: RNA ratio than ribosomes extracted from blastula stage ribosomes. Approximately 64 additional protein equivalents are found per ribosome. Most of the proteins are of high molecular weight and are tightly bound, being resistant to high-salt and EDTA treatment. The majority of the proteins appear to be basic in nature and remain associated with the 40S subunit on dissociation of the ribosomes. The possible physiological significance of the additional proteins is discussed in terms of the activation of protein synthesis following fertilisation. Sea urchin ribosomes, isolated from various stages of development, showed differential protein-labelling patterns. The high molecular-weight proteins had preferentially higher specific activities and one ribosomal protein was particularly highly labelled, reaching a maximum at the gastrula stage of development. The functional role of this highly labelled protein during development is discussed.