Neutrophils contact to plasma membrane of keratinocytes including desmosomal structures in canine pemphigus foliaceus

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that affects certain mammals including dogs. In canine PF, neutrophils are infiltrated intensely into pustular lesions including acantholytic cells, although neutrophilic infiltration is not characterized in human PF. The roles of the neutrophils in the cutaneous lesions of canine PF have not yet been understood. The purpose of this study was to characterize the ultrastructural features underlying the acantholysis with pustule formation in canine PF. Four dogs diagnosed as PF on the basis of clinical signs, histopathological findings, and direct and indirect immunofluorescence examinations were performed. Electron microscopy revealed that the acantholytic cells were adjacent to multiple neutrophils in the pustules. At the contact points between neutrophils and acantholytic keratinocytes, half-desmosomes of acantholytic keratinocytes with intact attachment plaques were observed within invaginations of neutrophils. Furthermore, on the surface of acantholytic cells in the pustules, neutrophil granules seemed to be secreted to the surface of acantholytic cells and to degenerate the half-desmosome structures. Neutrophils were also observed within the epidermis adjacent to the pustule. At the intercellular gap between two dissociated keratinocytes, neutrophils inserted its pseudopodia into the gap between the two half-desmosomes of keratinocytes. These findings taken together suggested that, at least in the areas where we analyzed ultrastructurally, neutrophils contact desmosomal structures and seem to play some parts in separation of keratinocytes and degeneration of split-desmosomes in pustules of dogs with PF.     

Expression of lipogenic genes during porcine intramuscular preadipocyte differentiation

Intramuscular fat (IMF) content plays an important role in meat quality. Triglyceride (TG) metabolism in intramuscular adipocytes is strongly associated with the intramuscular fat deposition. To better understand the mechanisms leading to IMF deposition we compared the expression levels of genes related to preadipocyte differentiation and lipogenesis in the intramuscular preadipocytes isolated from the longissimus muscle of Wujin and Landrace pigs. The results showed that the intramuscular preadipocytes could differentiate into mature adipocytes in vitro. Triglyceride content in adipocytes isolated from Wujin pigs was higher than Landrace pigs during the middle and later phases of preadipocyte differentiation. The expression levels of genes related to preadipocyte differentiation such as PPARG and CEBPA showed differential expression between Wujin and Landrace porcine adipocytes during the early stage of differentiation. The expression levels of lipogenic genes such as FASN and SREBF1 were significantly higher in Wujin porcine intramuscular preadipocytes than in Landrace intramuscular preadipocytes at the middle and the later stages of differentiation. This suggests that preadipocyte differentiation and lipogenesis exhibited breed-related scheduling.     

Expression of leptin mRNA and CCAAT-enhancer binding proteins in response to insulin deprivation during preadipocyte differentiation in primary cultures of porcine stromal-vascular cells

The aim of this study was to examine the correlation between CCAAT-enhancer binding proteins (C/EBPs) and leptin gene expression in response to insulin deprivation in preadipocytes and adipocytes. Adipose tissue from 7 d-old pigs was digested enzymatically and stromal-vascular (S-V) cells were seeded and plated for 3 d in fetal bovine serum (FBS) with dexamethasone (DEX) followed by 6 d (Days 3–9) in serum-free medium with insulin (850 nM or 10 nM), transferrin, and selenium. During FBS+DEX treatment (Days 0–3) a large number of preadipocytes develop with no lipid accretion. In contrast, preadipocyte number does not change with lipid accretion during insulin treatment (Days 3–9). Total RNA and cells were harvested from S-V cultures after periods with and without insulin after FBS+DEX. Northern-blotting and Western blot analysis were used to study leptin mRNA and C/EBP protein expression in cultures, respectively. Insulin deprivation from Days 3–4 reduced leptin mRNA and C/EBP- protein expression. Treatment with 850 nM or 10 nM insulin from Days 3–9 induced leptin mRNA and C/EBP- expression at a similar level. In cultures treated with 10 nM insulin from Days 3–7, leptin and C/EBP- expression were reduced markedly by insulin deprivation from Days 7–9, but were restored by insulin treatment for 6 hr before harvesting. The restoration of leptin expression by insulin was blocked by cycloheximide treatment. However, C/EBP-β protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7–9 in cultures treated with 850 nM insulin from Days 3–7 did not influence C/EBP- or leptin mRNA expression, whereas C/EBP- and leptin expression were reduced after treating these cultures with 1.5 uM okadaic acid for 45 min before harvesting on Day 9. However, cycloheximide treatment for 6 hr before harvesting did not reduce leptin mRNA expression. These results suggest that 1) leptin expression is positively correlated with C/EBP- expression, and 2) the maintenance of leptin expression after insulin deprivation in 850 nM insulin-treated cultures on Day 9 may be associated with the presence of C/EBP- expression and/or activation.     

Disruption of endogenous perlecan function improves differentiation of rat articular chondrocytes in vitro

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) are necessary for normal cartilage development and chondrocyte differentiation. However, recent studies demonstrated that HSPG accelerate dedifferentiation and catabolism in chondrocytes from degenerative cartilage. In this study, we investigated the inhibitory effect of HSPG on chondrocyte differentiation in vitro. Rat articular chondrocytes were cultured at low (0.3 × 10⁴ cells/cm²) and high (1.5 × 10⁵ cells/cm²) density in the presence or absence of heparitinase I, an HS degrading enzyme. Cells cultured at low density dedifferentiated and exhibited an elongated morphology, and treatment with heparitinase I precluded cell elongation. Conversely, populations of chondrocytes cultured at high density exhibited either a dedifferentiated or differentiated phenotype. Glycosaminoglycan accumulation increased in heparitinase I‐treated cells. To determine the function of perlecan, an important HSPG for cartilage development, in chondrocyte differentiation, rat chondrocyte cultures were exposed to an anti‐perlecan antiserum to inhibit perlecan function. Western blotting analysis indicated that preventing perlecan activity increased type II collagen synthesis. Our results suggest that HSPG are negative regulators of chondrocyte differentiation in vitro and that perlecan contributes to chondrocyte dedifferentiation in vitro.     

酪氨酸磷酸化蛋白在体外获能猪精子上的分布

为研究猪精子获能过程中蛋白酪氨酸磷酸化的变化规律,将猪精子悬浮于改良的Tris缓冲液(mTBM)获能培养基中,在5%CO2孵箱37℃培养,以考马斯亮蓝染液染色为指标评价精子获能状态,用免疫荧光技术检测酪氨酸磷酸化的蛋白在精子上的分布。结果显示,随着获能的进行,发生蛋白酪氨酸磷酸化的精子占总精子的百分比增加,由未获能前35%增至获能1.5 h时的88%,酪氨酸磷酸化的蛋白分布变广,由精子头部扩增至精子头部、鞭毛主段和中段。     

Expression of MAP kinases and connexins in the differentiation of rat mammary epithelial cells

Gap junctional intercellular communication (GJIC) is involved in the regulation of many cellular processes. MAP kinases are known to affect GJIC and phosphorylation of connexin (Cx). MAP kinases can also be a regulator of cell proliferation and growth. This study was undertaken to show the relevance between expression patterns of Cxs and MAP kinases in rat mammary epithelial cells (RMECs). In order to characterize the RMECs, they were stained with Peanut lectin, which indicates most alveolar epithelial cells, and Thy-1.1 was used as a marker of luminal epithelial cells or myoepithelial cells, respectively. We studied the expression patterns of major gap junction proteins, Cx26, 32, and 43 in RMECs. Western blot analysis demonstrated that Cx26 gradually decreased from day 2, while Cx32 was expressed constantly from day 1 to 14. Cx43 dramatically increased on day 5 and decreased thereafter. The expression patterns and phosphorylation of ERK1/2 and JNK were similar to Cx43, but expression of p38 was like that of Cx32. These results showed that the MAP kinases that comprise ERK1/2, p38, and JNK were involved in regulation of Cxs. Our data suggests that GJIC plays an important role during rat mammary differentiation and that MAP kinases may be closely related functionally to regulate the gap junction.     

Expression and localisation of Bcl-2 and Bax proteins in developing rat ovary

Bcl-2 and Bax proteins localised mainly in granulosa cells. Primordial and primary follicles of new born rat ovary showed an intensive nuclear staining for Bax but faint staining for Bcl-2. In terms of staining intensity, no remarkable difference was observed within the same stage of developing follicle. Compared to new born rats, granulosa cells of adult and one month old rat ovary showed an increased staining both for Bcl-2 and Bax proteins. No staining was observed in primordial follicles of one month old and adult rats. However, granulosa cells of primary follicles, granulosa cells and theca cells in tertiary follicles of adult rat ovary also showed a strong staining for Bcl-2 and Bax proteins. Oocytes of follicles from different developmental stages revealed an apparent staining both for Bcl-2 and Bax proteins. However, in the more mature follicles oocytes stained more intensively. In developing corpus luteum a remarkable staining was observed for Bax. However, the staining was more prominent in regressing corpus luteum. Contrary to this, Bcl-2 stained the luteal cells in developing corpus luteum strongly, while in the fully developed corpus luteum no staining for Bcl-2 was observed. In conclusion, there was an apparent relation between the expression of the apoptosis regulating protein Bcl-2 and Bax and follicular development. Thus, during the follicular development Bcl-2 and Bax may be involved in granulosa cell demise in rat ovary. Furthermore, increased levels of Bax and decreased levels of Bcl-2 in the fully developed corpus luteum suggest that Bax plays a role in apoptosis of luteal cells in rat ovary.     

组织型纤溶酶原激活剂基因在牛卵丘-卵母复合体体外成熟进程中的表达

为了初步了解组织型纤溶酶原激活剂(tissue-plasminogen activator,tPA)在牛卵丘－卵母复合体体外成熟进程中的潜在作用,试验运用实时定量RT-PCR技术分别检测体外成熟过程中不同时间段(0、8、16、24 h)以及添加不同浓度FSH成熟培养16 h后的牛卵丘细胞和卵母细胞中tPA基因相对表达变化,观察添加不同浓度FSH成熟培养16 h后卵丘细胞膨胀程度差异,并统计卵母细胞第一极体排出情况。结果显示,卵丘－卵母复合体在体外成熟初期,tPA mRNA相对表达水平在体外成熟16和24 h的卵丘细胞和卵母细胞中显著高于0和8 h组;在添加不同浓度FSH (0、0.01、0.1和1 IU/mL)体外成熟16 h的各处理组,卵丘细胞中tPA mRNA相对表达量随FSH添加浓度的升高而升高,卵丘细胞的扩散程度亦随之增加;同时tPA mRNA相对表达量在0.01 IU/mL FSH添加组卵母细胞中显著高于其他各FSH添加组。综合以上研究结果,本研究认为,牛卵丘细胞中tPA基因的表达受FSH正向调节;tPA可能促进了卵丘细胞在体外成熟过程中的扩散;同时,tPA在卵母细胞中的表达是否与其第一极体排出有关,需进一步试验验证。     

山羊GDF9基因在卵丘卵母细胞复合体体外成熟过程中的表达

本研究采用实时荧光定量PCR技术,从mRNA水平探讨了山羊卵丘卵母细胞复合体体外成熟(IVM)过程中(0、6、12、18、24和27h)GDF9基因的相对表达变化,分析其与卵丘细胞扩散之间的关系。结果表明,GDF9 mRNA在山羊卵丘卵母细胞复合体体外成熟过程中均有表达,且在成熟培养初期表达量较低,其表达峰值出现在IVM12h,与卵丘细胞开始扩散的时间一致,之后又有所下降。由此推测,GDF9基因在山羊卵丘扩散和卵母细胞体外成熟过程中起着重要的作用。     

Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro

Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.     

Expression of hedgehog family genes in the rat uterus during early pregnancy

Hedgehog (Hh) plays a pivotal role in various tissues during embryonic development, tissue homeostasis and tumorigenesis. In mammals, Hh exists in three homologs: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). In this study, we cloned full-length cDNAs encoding Dhh and Ihh from the rat uterus. Their amino acid sequences have a high homology with those of the mouse and human. In addition, the changes of Hh gene expression in the rat uterus during early pregnancy were analyzed. The results showed that all three hedgehog mRNAs were detected in the rat uterus at the proestrus stage and during early pregnancy (1.5, 3.5, 5.5 and 7.5 days post coitus: dpc). Ihh mRNA expression varied and peaked at 3.5 dpc in the luminal and glandular epithelium. Expression was decreased on 5.5 dpc with the exception of sustained expression in the glandular epithelium. Despite such Ihh variability, the expressions of Dhh and Shh mRNA remained unchanged. This indicated that Ihh was mainly expressed in the rat uterus during early pregnancy. Moreover, the Hh target gene (glioma-associated oncogene homolog 1; Gli1) was also highly expressed at 3.5 dpc in the epithelium and periepithelial stroma in a manner similar to the temporal pattern of Ihh expression. This suggests that Ihh signaling axis play a role in the rat uterus during early pregnancy. In summary, our results elucidate that Ihh is a predominant Hh protein in the rat uterus during early pregnancy and that other Hhs have the potential to be expressed. This observation will help to elucidate the basic molecular mechanism of rat uterus during early pregnancy.     

Expression of arginase by mouse myeloid leukemic cell differentiation in vitro induced with tumor necrosis factor.

Induction of arginase activity in mouse myeloid leukemic M1 cells by treatment with recombinant human tumor necrosis factor (rH-TNF) or TNF-elicited mouse serum (TNS) were examined in vitro. M1 cells differentiated into macrophage-like cells by addition of rH-TNF or TNS. The differentiated cells expressed phagocytic function and did not grow anymore. Cytolytic effect of rH-TNF or TNS was not observed. The differentiation of M1 cells into phagocytic cells by the TNS treatment was more rapidly than that by the rH-TNF treatment though TNS contained 25-time less amounts of TNF indicating species specificity of TNF action on myeloid leukemic cells or TNS containing other differentiation factor(s). 3H-ornithine formation from 3H-arginine is catalyzed by arginase (EC. 3.5.3.1). The enzyme product increased in the M1 cell culture medium by the treatment with rH-TNF or TNS. The arginase activity statistically correlated with the appearance percentage of differentiated cells with phagocytic function. These results suggest that in vitro differentiation of M1 cells is accompanied by induction of arginase activity.     

钙对体外培养大鼠成骨细胞增殖分化及细胞周期的影响

在体外培养大鼠成骨细胞的过程中,添加不同剂量的钙(0、1、2、4 mmol/L),通过检测细胞周期、细胞增殖能力、碱性磷酸酶(ALP)活性及骨桥蛋白表达,探讨了钙对成骨细胞增殖分化及细胞周期的影响.结果表明,2、4 mmol/L钙组在1～8 d均显著或极显著地促进成骨细胞增殖(P<0.05或P<0.01),1 mmol/L钙组则在5、6、8 d有显著或极显著的差异(P<0.05或P<0.01);添加不同浓度的钙除1 mmol/L组第2天外的不同时间均极显著地抑制细胞内ALP活性(P<0.01);1 mmol/L钙能极显著的使成骨细胞滞留在S期(P<0.01);添加不同浓度的钙均能使G2期显著或极显著的增加(P<0.05或P<0.01),G1期极显著的下降(P<0.01),并能极显著地诱导细胞内骨桥蛋白的表达(P<0.01).表明添加不同浓度的钙均能抑制其早期分化,促进成骨细胞的增殖,使细胞滞留在S期或G2期,诱导细胞在基质成熟期分化,有利于细胞的钙化.     

大鼠脂肪细胞分化过程中脂代谢相关基因转录表达时序的研究

对大鼠附睾和肾周脂肪组织分离的前体脂肪细胞原代培养，采用RT—PCR法分析脂代谢重要酶和转录因子基因在不同分化阶段转录表达的时序变化。结果显示，在前体脂肪细胞阶段，固醇调控元件结合蛋白（SREBP）-1c、碳水化合物反应元件结合蛋白（ChREBP）以及脂肪酸合成酶（FAS）、乙酰辅酶A羧化酶α（ACC1）、硬酯酰辅酶A去饱和酶（SCD）和激素敏感脂酶（HSL）基因均不转录表达；HSL mRNA在分化初期表达，中期表达水平最高，分化末期降低；SCD mRNA在分化中期表达，末期表达水平显著提高；分化初期FAS mRNA水平明显高于中期和末期；ACC1 mRNA仅在末期表达；分化初期SREBP-1c mRNA水平较高，中期和末期显著下降；ChREBP mRNA表达水平在分化末期稍高于中期，但差异不显著。表明，SREBP-1c mRNA的表达与脂肪细胞早期分化调控有关，分化成熟脂肪细胞的ChREBP mRNA表达可能与生脂基因的转录激活有关。     

Expression and regulation of cyclooxygenase-2 in normal and neoplastic canine keratinocytes

Squamous cell carcinoma (SCC) is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Increasing evidence implicates cyclooxygenase‐2 (COX‐2) in the pathogenesis of various cancers in humans and animals. COX‐2 overexpression has recently been demonstrated in canine SCCs. The objective of our study was to characterize the expression and regulation of COX‐2 in normal and neoplastic canine keratinocytes (CKs) to provide an in vitro system to investigate the implication of COX‐2 in SCC oncogenesis in dogs. Cell lines derived from normal CKs and neoplastic CKs (SCCs) were cultured in the absence or presence of agonists, and immunoblots, immunocytochemistry, radioimmunoassays and a cell proliferation assay were used to characterize COX‐2 expression and action. Results showed that neoplastic keratinocytes had a higher basal COX‐2 expression than normal keratinocytes. In both cell lines, stimulation with the tumour promoter phorbol 12‐myristate 13‐acetate induced a time‐dependent increase in COX‐2 protein, with COX‐2 induction being stronger in cancerous SCC than in normal CK cells. Moreover, SCC cells produced significantly more PGE₂ than CK cells, under both baseline and stimulated conditions (P < 0.05). NS‐398, a selective COX‐2 inhibitor, inhibited prostaglandin (PG)E₂ synthesis and decreased proliferation of CK and SCC cells (P < 0.05). Collectively, our results indicate that the canine neoplastic keratinocyte SCC cell line expresses more COX‐2 and produces more PGE₂ than the normal keratinocyte CK cell line, thus providing an in vitro system to study the molecular basis of elevated COX‐2 expression in SCCs in dogs.     

Expression and regulation of Foxa2 in the rat uterus during early pregnancy

The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.     

Expression and localization of tight junction-related proteins in adult rat pituitary stem/progenitor cell niches

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100β, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.     

Expression and regulation of Enpp2 in rat uterus during the estrous cycle

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (Enpp2) isolated from the supernatant of human melanoma cells is a lysophospholipase D that transforms lysophosphatidylcholine into lysophospatidic acid. Although multiple analyses have investigated the function of Enpp2 in the hypothalamus, its role in the uterus during the estrous cycle is not well understood. In the present study, rat uterine Enpp2 was analyzed by RT-PCR, Western blotting, and immunohistochemistry. Quantitative PCR analysis demonstrated that uterine Enpp2 mRNA was decreased during estrus compared to proestrus and diestrus. To determine whether uterine Enpp2 expression is affected by sex steroid hormones, immature rats were treated with 17β-estradiol (E2), progesterone, or both on postnatal days 14 to 16. Interestingly, the expression of Enpp2 mRNA and protein were down-regulated by E2 in the uterus during estrus but not during proestrus or diestrus, suggesting that Enpp2 may play a role in uterine function during estrus. Enpp2 is primarily localized in the stromal cells of the endometrium during proestrus and estrus. During diestrus, Enpp2 was highly expressed in the epithelial cells of the endometrium. Taken together, these results suggest that uterine Enpp2 may be regulated by E2 and plays a role in reproductive functions during female rat development.     

绵羊卵母细胞和卵丘细胞体外成熟过程中ghrelin mRNA的表达

运用实时定量RT-PCR技术检测了不同成熟培养时间绵羊卵母细胞和卵丘细胞ghrelin mRNA的相对表达量。结果表明,卵母细胞ghrelin mRNA相对表达量在16、24h呈现显著升高,而卵丘细胞ghrelin mRNA则随着成熟时间的延长表现显著降低。绵羊卵母细胞和卵丘细胞ghrelin mRNA的持续表达以及动力学变化提示这一新型分子在生殖功能上有潜在调控作用。