Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.     

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth

Adherent cells interact with extracellular matrix via cell–substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.     

Inhibitory role of polyunsaturated fatty acids on lysophosphatidic acid-induced cancer cell migration and adhesion

Polyunsaturated fatty acids (PUFAs) have important pharmacological effects on mammalian cells. Here, we show that carboxyl group-containing PUFAs inhibit lysophosphatidic acid (LPA)-induced focal adhesion formation, thereby inhibiting migration and adhesion. Carboxyl group-containing PUFAs inhibit LPA-induced calcium mobilization, whereas ethyl ester-group containing PUFAs have no effect. In addition, carboxyl group-containing PUFAs functionally inhibit LPA-dependent RhoA activation. Given these results, we suggest that PUFAs may inhibit LPA-induced calcium/RhoA signaling pathways leading to focal adhesion formation. Carboxyl group-containing PUFAs may have a functional role in this regulatory mechanism.     

Susceptibility to persistent breeding-induced endometritis in the mare: relationship to endometrial biopsy score and age, and variations between seasons

The objectives were to: (1) investigate the associations of age and endometrial biopsy score with uterine fluid retention after insemination; and (2) determine if a strict classification of susceptibility to persistent breeding-induced endometritis (PBIE) based on biopsy score, endometrial cytology, and fluid retention after inseminations, is consistent over subsequent breeding seasons. In Experiment 1, 57 mares were inseminated with 10⁹ freeze-killed sperm during estrus and evaluated for uterine fluid retention 48 h and 96 h after insemination. Comparisons were made between fluid retention and biopsy score or age. In Experiment 2, a subset of 14 mares was classified for susceptibility to persistent breeding-induced endometritis in two subsequent breeding seasons. Biopsy score and age were associated with fluid retention (P < 0.001). In addition, age was related to biopsy score (P < 0.001). Of the mares examined for susceptibility, 36% (5 of 14) changed status during subsequent seasons. Three mares changed to a more severe classification (intermediate to susceptible, or resistant to intermediate), whereas two mares changed to a less severe classification (susceptible to intermediate).     

Effect of intrauterine administration of gonadotropin releasing hormone on serum LH concentrations in lactating dairy cows

The objectives were to compare: (1) preovulatory serum LH concentrations, and (2) synchronization of ovulation, after im or iu administration of the second GnRH treatment of Ovsynch in lactating dairy cows. Lactating cows (N = 23) were presynchronized with two injections of PGF_2α given 14 days apart (starting at 34 ± 3 days in milk), followed by Ovsynch (GnRH-7 d-PGF_2α-56 h-GnRH) 12 days later. At the time of the second GnRH of Ovsynch (Hour 0), cows were blocked by parity and randomly assigned to 1 of 3 groups: (1) control group (CON; N = 7) were given 2 mL sterile water im; (2) intramuscular group (IM; N = 8) received 100 μg of GnRH im; and (3) intrauterine group (IU; N = 8) had 100 μg GnRH infused in the uterus (2 mL). Blood samples for serum LH concentrations were collected at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Furthermore, ultrasonography was performed twice daily (12-h intervals) from Hours 0 to 60 to confirm ovulation. The LH concentrations were greater (P < 0.05) in the IM than IU and CON groups at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Although LH concentrations were numerically higher in the IU group, LH concentrations within the IU and CON groups did not change over time. More cows ovulated in the IM (8/8) and IU (7/8) groups within 60 h after the second GnRH administration compared with the CON (2/7) group. In summary, serum LH concentrations were lower in the IU versus IM group, but the proportion of cows that ovulated within 60 h was similar between these two groups. Therefore, iu administration of GnRH may be an alternative route of delivery to synchronize ovulation in beef and dairy cattle.     

逆行射精精子冷冻保存在宫腔内人工授精技术中的应用

目的:探讨逆行射精患者尿液中回收精子的冷冻保存及其在宫腔内人工授精(IUI)周期中的应用效果。方法:2008年12月至2011年1月逆行射精患者共计7例,嘱其按要求留取射精后的尿液,充分洗涤后实施液氮蒸汽法超低温精子冷冻,共冻存精液标本14人份。IUI周期时将冻存精子解冻、优化后行宫腔内人工授精。结果:冷冻前、解冻后前向运动精子总数分别为(19.9±10.4)×106和(6.9±4.2)×106,存在显著性差异(P<0.05),解冻优化后前向运动精子总数为(2.6±1.7)×106,实施IUI 8个周期,临床妊娠1例。结论:超低温冷冻会明显降低前向运动精子总数,但多次冷冻后一次复苏行IUI可能是治疗逆行射精所致不育的一种较为理想的方法。     

Nonlinear mechanical modeling of cell adhesion

Cell adhesion, which is mediated by the receptor-ligand bonds, plays an essential role in various biological processes. Previous studies often described the force-extension relationship of receptor-ligand bond with linear assumption. However, the force-extension relationship of the bond is intrinsically nonlinear, which should have significant influence on the mechanical behavior of cell adhesion. In this work, a nonlinear mechanical model for cell adhesion is developed, and the adhesive strength was studied at various bond distributions. We find that the nonlinear mechanical behavior of the receptor-ligand bonds is crucial to the adhesive strength and stability. This nonlinear behavior allows more bonds to achieve large bond force simultaneously, and therefore the adhesive strength becomes less sensitive to the change of bond density at the outmost periphery of the adhesive area. In this way, the strength and stability of cell adhesion are soundly enhanced. The nonlinear model describes the cell detachment behavior better than the linear model.     

Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus

Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection. In this study, we selected newborn infants of HBsAg-positive mothers, and divided the infants into 2 groups: intrauterine infection group and non-intrauterine infection group according to the status whether or not they were infected at birth. Each infected infant was compared with 2 controls from the same birth cohort. HLA-DR allele typing was performed using a PCR-sequence specific primer (PCR-SSP) for 24 subjects with intrauterine infection and 48 controls without infection. We found that, among the fifteen (15) HLA-DR alleles assessed, HLA-DRB1*07 was the one, and the only one, significantly in excess (OR = 6.66, P = 0.004) in the intrauterine infection group compared to the non-intrauterine infection group. Our findings thus suggest that high frequency of HLA class II molecules, e.g. HLA-DRB1*07, is associated with the susceptibility of the infants to intrauterine HBV infection.     

人羊膜上皮干细胞联合富血小板血浆对宫腔粘连大鼠纤维化因子、炎症因子的作用

摘要 目的：探究人羊膜上皮干细胞（hAECs）联合富血小板血浆（PRP）对宫腔粘连大鼠的治疗效果,为临床中优化宫腔粘连治疗方法理论基础。方法：选用SPF级雌性SD大鼠共40只,依照随机数字表法随机分为空白组（A组）、假手术组（B组）、hAECs组（C组）、PRP组（D组）、hAECs+PRP组（E组）,每组各8只。A组不做任何处理,B组仅做麻醉、开关腹腔处理,C组、D组、E组使用搔刮法制备宫腔粘连模型。随后C组进行hAECs治疗,D组注射等量生理盐水。D组进行PRP治疗,C组注射等量生理盐水。E组进行hAECs联合PRP治疗。观察比较两组大鼠子宫内膜组织基质金属蛋白酶-9（MMP-9）、盘状结构域受体2（DDR2）蛋白表达水平、PI3K/Akt/mTOR通路相关mRNA表达量、血清白细胞介素-6（IL-6）、白细胞介素-8（IL-8）水平以及各组大鼠妊娠孕囊数量比较。结果：与A组相比,B组的子宫内膜组织MMP-9、DDR2蛋白表达水平、p-mTOR mRNA、p-Akt mRNA水平以及血清IL-6、IL-8水平比较无差异（P＞0.05）;C组、D组、E组大鼠的子宫内膜组织MMP-9、DDR2蛋白表达水平明显下降,IL-6、IL-8水平、p-mTOR mRNA、p-Akt mRNA表达升高（P＜0.05）。其中,E组大鼠MMP-9、DDR2蛋白表达水平明显高于C组、D组,IL-6、IL-8水平及p-mTOR mRNA、p-Akt mRNA表达低于C组、D组（P＜0.05）。C组、D组大鼠MMP-9、DDR2蛋白表达水平、血清IL-6、IL-8水平及p-mTOR mRNA、p-Akt mRNA表达比较（P＞0.05）。妊娠孕囊数量方面,与A组比较,B组大鼠妊娠孕囊数量。结论：hAECs联合PRP治疗可能通过上调宫腔粘连大鼠子宫内膜组织MMP-9、DDR2蛋白表达水平并下调p-mTOR mRNA、p-Akt mRNA表达,同时降低炎性因子IL-6、IL-8水平,进而提高妊娠孕囊数量,起到治疗作用。     

6-C-kine (SLC), a Lymphocyte Adhesion-triggering Chemokine Expressed by High Endothelium, Is an Agonist for the MIP-3β Receptor CCR7

The β chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related β chemokine MIP-3β (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3β attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3β inhibits lymphocyte chemotaxis to 6CK/SLC but not to the α chemokine SDF-1 (stromal cell–derived factor); and 6CK/SLC competes for MIP-3β binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3β in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.