Mechanisms of sperm-egg interactions emerging from gene-manipulated animals

Untangling the molecular nature of sperm-egg interactions is fundamental if we are to understand fertilization. These phenomena have been studied for many years using biochemical approaches such as antibodies and ligands that interact with sperm or with eggs and their vestments. However, when homologous genetic recombination techniques were applied, most of the phenotypic factors of the gene-manipulated animals believed “essential” for fertilization were found to be dispensable. Of course, all biological systems contain redundancies and compensatory mechanisms, but as a whole the old model of fertilization clearly requires significant modification. In this review, we use the results of gene manipulation experiments in animals to propose the basis for a new vision. Received 26 January 2007; received after revision 7 March 2007; accepted 17 April 2007     

Receiving mixed signals: uncoupling oligodendrocyte differentiation and myelination

The development and maturation of an oligodendroglial cell is comprised of three intimately related processes that include proliferation, differentiation, and myelination. Here we review how proliferation and differentiation are controlled by distinct molecular mechanisms and discuss whether differentiation is merely a default of inhibited proliferation. We then address whether differentiation and myelination can be uncoupled in a similar manner. This task is particularly challenging because an oligodendrocyte cannot myelinate without first differentiating, and these processes are therefore not mutually exclusive. Is it solely the presence of the axon that distinguishes a differentiated oligodendrocyte from a myelinating one? Uncoupling these two processes requires identifying specific signals that regulate myelination without affecting the differentiation process. We will review current understanding of the relationship between differentiation and myelination and discuss whether these two processes can truly be uncoupled.     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis

Cancer cells are typically characterized by apoptosis deficiency. In order to investigate a possible role for the anti-apoptotic livin gene in renal cell cancer (RCC), we analyzed its expression in tumor tissue samples and in RCC-derived cell lines. In addition, we studied the contribution of livin to the apoptotic resistance of RCC cells by RNA interference (RNAi). Livin gene expression was detected in a significant portion of RCC tumor tissue specimens (13/14, 92.9%) and tumor-derived cell lines (12/15, 80.0%). Moreover, targeted inhibition of livin by RNAi markedly sensitized RCC cells towards proapoptotic stimuli, such as UV irradiation or the chemotherapeutic drugs etoposide, 5-fluorouracil, and vinblastine. These effects were specific for livin expressing tumor cells. We conclude that livin can contribute significantly to the apoptosis resistance of RCC cells. Targeted inhibition of livin could represent a novel therapeutic strategy to increase the sensitivity of renal cancers towards pro-apoptotic agents. Received 30 November 2006; received after revision 22 February 2007; accepted 20 March 2007     

The peptidoglycan recognition proteins LCa and LCx

Infection of bacteria triggers innate immune defense reactions in Drosophila. So far, the only bacterial component known to be recognized by the insect innate immune system is peptidoglycan, one of the most abundant constituents of the bacterial cell wall. Insects use peptidoglycan recognition proteins to detect peptidoglycan and to activate innate immune responses. Such specialized peptidoglycan receptors appear to have evolved from phage enzymes that hydrolyze bacterial cell walls. They are able to bind specific peptidoglycan molecules with distinct chemical moieties and activate innate immune pathways by interacting with other signaling proteins. Recent X-ray crystallographic studies of the peptidoglycan recognition proteins LCa, and LCx bound to peptidoglycan have provided structural insights into recognition of peptidoglycan and activation of innate immunity in insects. Received 28 December 2006; received after revision 2 February 2007; accepted 21 February 2007     

Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive. Received 3 September 2007; received after revision 17 October 2007; accepted 22 October 2007     

The human genome and understanding of common disease: present and future technologies

The study of candidate genes over the past three decades has yielded notable successes in common-disease genetics. During this time, however, interpretation of genetic association studies has been hampered by the use of clinical cohorts of inadequate power and insufficient information on genetic variation in candidate genes. The unavailability of highthroughput and low-cost genotyping technologies has also limited the scope of complex-disease genetic studies. More recently, however, the sequencing and characterization of variation within the human genome has revolutionized genetic studies and enabled full genome-wide scans for genes associated with disease. The identification of disease-associated (causative) genes has illuminated disease mechanisms. The translation of this knowledge into direct clinical benefit in diagnosis, prognosis and therapy for an individual’s disease still remains a challenge. Received 11 September 2006; received after revision 17 December 2006; accepted 18 January 2007