Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.     

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background  

The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).     

Zur Genetik der progressiven Myoklonusepilepsien (Unverricht-Lundborg)

Ohne ZusammenfassungMit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway

A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

犬MC1R基因T105A基因座多态性及 与毛色性状相关性的研究The

为了检测犬MC1R基因T105A基因座的多态性,并分析该多态性与犬毛色表型的相关性,抽取111只外科手术学实验用杂种犬血液并提取DNA,记录毛色表型。采用PCR-RFLP技术,对MC1R基因T105A基因座进行基因多态性分析,并对该基因座DNA进行克隆测序;用二元变量相关分析的统计学方法分析基因座多态性与毛色性状之间的相关性。经PCR-RFLP分析结果表明,T105A基因座序列具有多态性,表现为A、B二个等位基因和AA、AB及BB 3种基因型。A、B等位基因频率分别为72.97%和27.03%,基因杂合度（H）为0.39。基因型AA频率为55.86%,BB为9.91%,AB为34.23%。对T105A多态性片段DNA克隆测序后发现,MC1R基因在编码第105位氨基酸的密码子第一个碱基存在由G到A的单碱基突变,该突变导致第105位氨基酸发生由丙氨酸向苏氨酸的改变。统计分析结果表明MC1R基因T105A基因座的多态性与毛色性状不存在显著的相关性,这可能是由于外科手术学实验用犬是杂种犬,其遗传背景不同所致,尚须在纯种犬群体中进一步研究MC1R基因对毛色的影响。 Abstract: In order to detect the polymorphism of T105A in MC1R gene in dogs and to analyze the relationship between the genetic polymorphisms and phenotypes of dog coat color, the blood samples of 111 cross-breed dogs were taken and their genomic DNAs were extracted. The phenotypes of dog coat color were recorded. The T105A locus of MC1R gene in the canine was detected through the technology of PCR-RFLP. Furthermore, the polymorphic fragments at T105A were sequenced. The relationships between the polymorphism of T105A and coat color trait were analyzed by the statistical methods of bivarate correlation analysis. By the method of PCR-RFLP, the T105A polymorphism was found with two alleles A and B and three genotypes AA, AB and BB. The frequencies of two alleles were 72.97% and 27.03%, respectively. The heterozygosity of T105A locus was 0.39. The frequencies of three genotypes were 55.86%, 34.23% and 9.91%, respectively. According to the results of sequencing, one base change from G to A at the position 105 was found at T105A locus and it altered amino acid at the position 105 from alanine to threonine. According to the statistical analysis, no significant association between the polymorphism of MC1R gene and the coat color was found and the result may be due to the differences of genetic background. Further research on MC1R gene should be done in pure breed dogs.