Cyclin ET, a new splice variant of human cyclin E with a unique expression pattern during cell cycle progression and differentiation.

Cyclin E is the regulatory subunit of the cdc2-related protein kinase cdk2 and is a rate limiting factor for the entry into S phase. To date, cyclin E is the only cyclin for which alternative splicing has been described. We report here the isolation of a new splice variant of cyclin E, termed cyclin ET, which has an internal deletion of 45 amino acids compared with the full-length cyclin E protein. Even though cyclin ETcontains an intact cyclin box, it is unable to complement a triple cln mutant strain of Saccharomyces cerevisiae or to interfere with rescue by cyclin E, indicating that an intact cyclin box is functionally insufficient. The expression pattern of cyclin ET during cell cycle entry, progression and differentiation differs from that of cyclin E. Thus, ET expression precedes that of the other isoforms during the G0-->S progression; it shows a sharp peak in early G1 in cells released from a mitotic block and is strongly down-regulated in terminally differentiated myeloid cells. These observations point to different functions for cyclin ET and E and show for the first time that the alternative splicing of cyclin E is a regulated mechanism governed by the cell cycle and differentiation.     

Antigen-presenting cells and anti-Salmonella immunity.

The present article summarizes studies aimed at addressing the role of antigen-presenting cell populations, particularly dendritic cells (DC), in the immune response to Salmonella typhimurium. Data from in vitro studies shed light on presentation of antigens expressed in Salmonella on major histocompatibility complex class I and class II molecules by infected DC and macrophages, and the activation state of DC following infection. Finally, data from in vivo studies addressing the role of DC and defined DC subsets during the host response to Salmonella using a murine infection model are discussed.     

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.     

Tolerization against atherosclerosis using heat shock protein 60

Atherosclerosis is a chronic inflammatory disease of the artery wall, and both innate and adaptive immunity play important roles in the pathogenesis of this disease. In several experimental and human experiments of early atherosclerotic lesions, it has been shown that the first pathogenic event in atherogenesis is intimal infiltration of T cells at predilection sites. These T cells react to heat shock protein 60 (HSP60), which is a ubiquitous self-antigen expressed on the surface of endothelial cells (ECs) together with adhesion molecules in response to classical risk factors for atherosclerosis. When HSP60 is expressed on the EC surface, it can act as a “danger-signal” for both cellular and humoral immune reactions. Acquired by infection or vaccination, beneficial protective immunity to microbial HSP60 and bona fide autoimmunity to biochemically altered autologous HSP60 is present in all humans. Thus, the development of atherosclerosis during aging is paid by the price for lifelong protective preexisting anti-HSP60 immunity by harmful (auto)immune cross-reactive attack on arterial ECs maltreated by atherosclerosis risk factors. This is supported by experiments, which shows that bacterial HSP60 immunization can lead and accelerate experimental atherosclerosis. This review article presents accumulating proof that supports the idea that tolerization with antigenic HSP60 protein or its peptides may arrest or even prevent atherosclerosis by increased production of regulatory T cells and/or anti-inflammatory cytokines. Recent data indicates that HSP60, or more likely some of its derivative peptides, has immunoregulatory functions. Therefore, these peptides may have important potential for being used as diagnostic agents or therapeutic targets.     

Mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation.

The biochemical interactions between the Cdk2/Cyclin E kinase and its inhibitor p27, were investigated using purified, recombinant p27 and CAK-phosphorylated Cdk2/Cyclin E. From kcat/Km determinations using either histone H1 or pRb as substrates, we found that Cdk2/Cyclin E has 60-fold higher specificity for pRb than for histone H1. The IC50 value of p27 increased with increasing Cdk2/Cyclin E concentrations while it remained constant at various ATP and histone H1 concentrations, suggesting that p27 acts as a tight binding inhibitor of Cdk2/Cyclin E. We also found that p27 could be phosphorylated by Cdk2/Cyclin E only at high enzyme concentrations, and that p27 forms a stable interaction with Cdk2/Cyclin E regardless of its phosphorylation state. Our results further indicate that the Cdk2/Cyclin E/p27 ternary complex is kinetically inactive as an enzyme; instead it serves as a substrate for Cdk2/Cyclin E. These results suggest that if phosphorylation of p27 by Cdk2/Cyclin E is involved in its ubiquitin-dependent degradation, as previously suggested, then the target for such event is the phosphorylated p27 bound to Cdk2/Cyclin E and not free p27.