Requirement for Rho in Integrin Signalling

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.     

Effects of wind erosion and sand burial on water relations and photosynthesis in Alhagi sparsifolia in the southern edge of the Taklimakan Desert

塔克拉玛干沙漠南缘风沙活动十分频繁, 风蚀和沙埋是该地区自然植被生长发育的重要影响因子。该文以塔克拉玛干沙漠南缘策勒绿洲-沙漠过渡带为研究区, 以该区域主要建群种植物骆驼刺(Alhagi sparsifolia)为研究对象, 对一次强沙尘天气过后沙丘表面5种不同风蚀沙埋状况的骆驼刺植物进行标定(包括10 cm风蚀、5 cm风蚀、不蚀不积、10 cm沙埋、30 cm沙埋), 天晴后测定其叶水势、叶片含水量、光合参数和叶绿素荧光等参数, 分析研究自然环境条件下风蚀和沙埋对骆驼刺水分和光合作用的影响。结果表明: (1)风蚀显著降低了骆驼刺叶水势和叶片含水量, 进而导致植物气孔导度降低, 并引起植物光合速率和蒸腾速率的下降。风蚀的植物水分利用效率低于沙埋, 特别是在10 cm风蚀深度明显降低。 (2)沙埋增加了骆驼刺的叶水势、叶片含水量和气孔导度, 并引起植物光合速率和蒸腾速率的上升, 水分利用效率也得到提升。(3)风蚀条件下骆驼刺所受胁迫增加, 但可以通过增加活性反应中心的数量和光化学效率来抵消胁迫造成的不利影响。沙埋条件下骆驼刺受胁迫减轻, 反应中心吸收的光能和用于光化学反应的能量随着沙埋程度增加而减小, 这是骆驼刺适应风沙环境的一种生存策略。(4)与5 cm风蚀以及10 cm沙埋相比, 10 cm风蚀显著抑制骆驼刺的生长, 30 cm沙埋则会显著促进骆驼刺的生长。     

小鼠胰腺大部分切除后再生集中区来源的初步探索

目的:探讨胰腺在某些损伤或病理条件下,由于细胞活跃增殖产生再生集中区域的细胞来源。方法:将27只成年ICR系小鼠分为9组,每组3只,其中1组进行假手术,其余8组进行小鼠胰腺大部分切除,分别在切除后12h,24h、36h、48h、3d、5d、7d、10d取材及冰冻切片,采用H-E染色、免疫荧光染色方法检测损伤后各时间段胰腺组织的形态变化和细胞增殖率。结果:H-E染色发现,胰腺手术72h后,剩余胰腺中就出现由细胞角蛋白阳性导管样结构组成的再生集中区,此区域细胞随后分化为功能性细胞类型,10d后消失检测不到。对胰腺再生集中区的定位研究表明,它们仅出现于切除后的伤口边缘。BrdU标记表明,胰腺再生集中区为细胞快速增殖区域,其出现与总导管增殖率提高同时发生,主/大导管和小导管增殖率上升都晚于再生集中区的出现。结论:小鼠胰腺大部分切除后再生集中区可能来源于腺泡细胞的快速增殖,而不是经由总-主/大-小导管-快速增殖区这一途径引起的来源于导管上皮细胞。     

降雨和汇流对黑土区坡面土壤侵蚀的影响试验研究

东北黑土区上坡汇流对坡面土壤侵蚀有重要影响,因此辨析降雨和汇流对黑土区坡面土壤侵蚀的影响对农田土壤侵蚀防治有重要意义。通过设计不同降雨强度和汇流速率以及二者组合的模拟降雨及上方汇流试验,分析了降雨和汇流对黑土坡面侵蚀的影响及其贡献。试验处理包括两个降雨强度(50 mm/h和100 mm/h)、两个汇流速率(50 mm/h和100 mm/h,即:10 L/min和20 L/min)、以及4种不同降雨强度和汇流速率的组合((50+50)mm/h、(50+100)mm/h、(100+50)mm/h和(100+100)mm/h)。结果表明,在50 mm/h和100 mm/h上方汇流引起的坡面侵蚀量仅分别是50 mm/h和100 mm/h降雨引起坡面侵蚀量的1.9%和0.6%;当降雨强度和坡上方汇流速率分别由50 mm/h增加至100 mm/h时,降雨试验处理下的坡面侵蚀量增加6.1倍,汇流试验处理下的坡面侵蚀量增加3.2倍,说明降雨对坡面土壤侵蚀的影响显著大于汇流的作用。在降雨和汇流组合试验中,总供水强度(降雨强度+汇流速率)为150 mm/h时,降雨强度为100 mm/h和汇流速率为50 mm/h组合试验的坡面侵蚀量是降雨强度为50 mm/h和汇流速率为100 mm/h组合试验坡面侵蚀量的7.9倍。在相同汇流条件下,降雨强度由50 mm/h增加到100 mm/h时,降雨强度的增加对坡面侵蚀量的贡献率为89.6%-99.5%;而在相同降雨条件下,坡面汇流速率由50 mm/h增加100 mm/h时,汇流速率的增加对坡面侵蚀量的贡献率为17.2%-78.7%,说明在东北黑土区防治坡面汇流对坡面土壤侵蚀影响也尤为重要。     

降雨能量对东北典型黑土区土壤溅蚀的影响

溅蚀特征研究可揭示溅蚀发生机理,而现有研究大多用溅蚀量来表征溅蚀特征,不能全面准确地反应溅蚀作用过程。为此,基于改进的试验土槽进行室内模拟降雨试验,研究降雨能量对坡面不同方向溅蚀量及溅蚀过程的影响。试验设计包括2种降雨强度(50 mm/h和100 mm/h)和10个降雨能量,其中10个降雨能量是通过2种降雨强度(50 mm/h和100 mm/h)和5个雨滴降落高度(3.5,5.5,7.5,9.5、11.5 m)来实现的。结果表明:在相同降雨强度下,坡面总溅蚀分量均随降雨能量的增加而增大。次降雨坡面溅蚀量均为向下坡最大,其次为侧坡溅蚀量,而向上坡溅蚀量最小。当降雨强度由50mm/h增加至100mm/h时,坡面向上坡溅蚀量增加2.3—5.0倍,向下坡溅蚀量增加1.7—5.1倍,侧坡溅蚀量增加1.9—4.3倍,总溅蚀量增加1.9—4.5倍,净溅蚀量增加1.2—6.4倍。对于不同降雨能量处理,坡面溅蚀率均表现为坡面产流前随降雨历时的增加而递增,产流后迅速达到峰值,之后逐渐减小并趋于稳定。定量分析了各溅蚀分量、总溅蚀量、净溅蚀量与降雨能量的关系,提出了溅蚀发生的降雨能量阈值,发现雨滴溅蚀发生的临界能量为3—6 J m~(-2)mm~(-1),且向上坡溅蚀量,向下坡溅蚀量,净溅蚀量和总溅蚀量皆与降雨能量呈幂函数关系,而侧坡溅蚀量与降雨能量呈二次多项式关系。     

Beta1 integrins regulate mammary gland proliferation and maintain the integrity of mammary alveoli

Integrin-extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of beta1 integrin in the mammary gland, Itgbeta1(flox/flox) mice were crossed with WAPiCre transgenic mice, which led to specific ablation of beta1 integrin in luminal alveolar epithelial cells. In the beta1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell-basement membrane associations. Activity of focal adhesion kinase (FAK) was also decreased in mutant mammary glands. Luminal cell proliferation was strongly inhibited in beta1 integrin mutant glands, which correlated with a specific increase of p21 Cip1 expression. In a p21 Cip1 null background, there was a partial rescue of BrdU incorporation, providing in vivo evidence linking p21 Cip1 to the proliferative defect observed in beta1 integrin mutant glands. A connection between p21 Cip1 and beta1 integrin as well as FAK was also established in primary mammary cells. These results point to the essential role of beta1 integrin signaling in mammary epithelial cell proliferation.     

Neuroprotective effect of N-acetyl-aspartyl-glutamate in combination with mild hypothermia in the endothelin-1 rat model of focal cerebral ischaemia

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.     

Inactivation of tensin3 in mice results in growth retardation and postnatal lethality

Tensin family is a group of focal adhesion proteins that interact with integrins, actin, and phosphotyrosine-containing proteins. To explore the in vivo functions of a new member of the family, tensin3, we have generated mutant mice with a disrupted tensin3 gene. Inactivation of tensin3 resulted in growth retardation and postnatal lethality in one third of the homozygous mutants. Histological analysis of those mutants showed incomplete development of the small intestine, lung, and bone. Villus formation in the small intestine was affected and cells migrated slower in the runt mutants. Their lungs also displayed enlarged air space suggesting defects in alveogenesis. In addition, the resting zone was thicker and fewer proliferating cells were present in the growth plates of tensin3(-/-) tibiae. These observations indicate that tensin3 is essential for normal development and functions of the small intestine, lung, and bone. These phenotypes of the runt tensin3(-/-) mice are similar to some clinical features of Silver-Russell syndrome (SRS) which is a genetically inherited defect. About 10% of SRS cases have been linked to abnormality in chromosome 7p11.2-13, where human tensin3 gene is located, suggesting a potential link between tensin3 and SRS.     

Controlled release from triple layer,donut-shaped tablets with enteric polymers

The purpose of this research was to evaluate triple layer, donut-shaped tablets (TLDSTs) for extended release dosage forms. TLDSTs were prepared by layering 3 powders sequentially after pressing them with a punch. The core tablet consisted of enteric polymers, mainly hydroxypropyl methylcellulose acetate succinate, and the bottom and top layers were made of a water-insoluble polymer, ethyl cellulose. Drug release kinetics were dependent on the pH of the dissolution medium and the drug properties, such as solubility, salt forms of weak acid and weak base drugs, and drug loading. At a 10% drug loading level, all drugs, regardless of their type or solubility, yielded the same release profiles within an acceptable level of experimental error. As drug loading increased from 10% to 30%, the drug release rate of neutral drugs increased for all except sulfathiazole, which retained the same kinetics as at 10% loading. HCl salts of weak base drugs had much slower release rates than did those of neutral drugs (eg, theophylline) as drug loading increased. The release of labetalol HCl retarded as drug loading increased from 10% to 30%. On the other hand, Na salts of weak acid drugs had much higher release rates than did those of neutral drugs (eg, theophylline). Drug release kinetics were governed by the ionization/erosion process with slight drug diffusion, observing no perfect straight line. A mathematical expression for drug release kinetics (erosion-controlled system) of TLDSTs is presented. In summary, a TLDST is a good design to obtain zero-order or nearly zero-order release kinetics for a wide range of drug solubilities.     

Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events

Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu-1, binds to collagen type IV (Coll IV) through beta1-integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450-457). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu-1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu-1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu-1 cells with 60 microM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu-1 cell cytoskeleton integrity by 1-5 muM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19 - 6.6 x 10(7)) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation but activated the ERKs in a dose dependent manner. beta1-integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/beta1-integrin mediated signaling events in Calu-1 cells, which involve FAK, ERKs, and PKC.