Possible role of extracellular nucleotides in ectopic ossification of human spinal ligaments

To reveal the involvement of extracellular nucleotides in the ossification process in ossification of the posterior longitudinal ligament of the spine (OPLL), the mRNA expression profiles of P2 purinoceptors, mechanical stress-induced ATP release, and ATP-stimulated expression of osteogenic genes were analyzed in ligament cells derived from the spinal ligament of OPLL patients (OPLL cells) and non-OPLL cells derived from the spinal ligaments of cervical spondylotic myelopathy patients as a control. The extracellular ATP concentrations of OPLL cells in static culture were significantly higher than those of non-OPLL cells, and this difference was diminished in the presence of ARL67156, an ecto-nuclease inhibitor. Cyclic stretch markedly increased the extracellular ATP concentrations of both cell types to almost the same level. P2Y1 purinoceptor subtypes were intensively expressed in OPLL cells, but only weakly expressed in non-OPLL cells. Not only ATP addition but also cyclic stretch raised the mRNA levels of alkaline phosphatase and osteopontin in OPLL cells, which were blocked by MRS2179, a selective P2Y1 antagonist. These increases in the expression of osteogenic genes were not observed in non-OPLL cells. These results suggest an important role of P2Y1 and extracellular ATP in the progression of OPLL stimulated by mechanical stress.     

Positioning of actin filaments and tension generation in skinned muscle fibres released after stretch beyond overlap of the actin and myosin filaments

Summary Skinned fibres from frog semitendinosus muscle were stretched in relaxing solution from a sarcomere length of 2.5 m to greater sarcomere lengths, and then shortened back to the original length. Fibres could be stretched up to sarcomere lengths of 3.3 m, and reshortened fully. If the original stretch was to a sarcomere length greater than 3.3 m, the extent of recovery was dependent on the magnitude of the stretch and the number of times the stretch/shorten cycle was repeated. When the original stretch was to sarcomere lengths beyond overlap of the thick and thin filaments, the thin filaments did not re-enter the thick filament array but buckled at the A-I junction. If these fibres were subsequently activated and contracted, the thin filaments re-entered the thick filament array, taking up approximately their former positions, and allowing reduced development of isometric tension.Finally, the present observations support the view that calcium-induced interactions of actin and myosin filaments in the presence of ATP play an important role in the organization of myofibrillar structure during differentiation (compare Hayashiet al. 1977; Shimada & Obinata, 1977).     

Activities of creatine kinase isoenzymes in single skeletal muscle fibres of trained and untrained rats

Biochemical changes in the creatine kinase isoenzyme compositions in single muscle fibres of different types in rats were induced by endurance running training. Single muscle fibres were dissected from the soleus and extensor digitorum longus muscles of Wistarstrain male rats trained on a motor-driven treadmill for 16 weeks. Each fibre was typed histochemically (SO, slow-twitch oxidative; FOG, fast-twitch oxidative glycolytic; FG, fast-twitch glycolytic), and the activities of total creatine kinase and its four isoenzymes (CK-MM, -MB,-BB, and mitochondrial creatine kinase) were measured. The endurance training did not affect the total creatine kinase activity, but resulted in significantly increased activities of CK-MB and CK-BB in SO and FOG fibres, and the mitochondrial enzyme activity in FOG and FG fibres. Endurance training induced biochemical changes in the isoenzyme compositions, specifically in FOG fibres. These results suggest that changes in creatine kinase isoenzymes with endurance training reflect changes in the energy metabolism in the different muscle fibres, supporting the hypothesis that the different isoenzymes play different roles in energy transduction.     

Alterations of Integrin Expression in Human Lung Cancer

Integrins are cell-surface receptors which are involved in cell-matrix and/or cell-cell adhesion. They have been suggested to play a role in tumor invasion and metastasis. We examined the expression of various integrin subunits in normal and cancer cells of the lung using 33 human lung cancer cell lines as well as 6 lung cancer samples from which tumor cell lines could be established. This study clearly demonstrated that changes in the expression of certain integrins occur frequently in lung cancer, especially in small cell lung cancer. Loss of the α1 subunit of the β₁ integrin family appears to be the most prominent change, although loss of other integrin subunits such as α₂ or emergence of some integrin subunits such as α_v can also be observed. These results suggest that changes in integrin expression may contribute to the invasive and/or metastatic behavior of lung cancer.     

Two waves of memory B-cell generation in the primary immune response

Memory B cells can be generated independently of germinal center (GC) formation and affinity maturation in Bcl-6-deficient mice, but the contribution of the GC-independent pathway for memory B-cell generation in normal mice remains unknown. To examine this, we administrated anti-inducible co-stimulator (ICOS) mAbs into mice at the onset of GC formation in the primary response. This manipulation affected the generation of GC B cells in the spleen, but neither IgG1 memory B cell nor production of IgG1 long-term antibody was affected. In ICOS-manipulated mice, GC B cells accumulated somatic mutations in the IgV(H) genes and underwent affinity maturation; however, memory B cells scarcely accumulated mutations and reconstituted the secondary response by low affinity, supporting the notion that low-affinity memory B cells are generated in a GC-independent manner. Thus, it appears that memory B cells are established by two different pathways, associated with or without GC reaction and affinity maturation. The generation and long-term persistence of low-affinity IgG1 memory B cells and antibodies in ICOS-manipulated mice support the idea that low-affinity memory B cells may give rise to long-term antibody-forming cells.     

Identification of human minor histocompatibility antigens based on genetic association with highly parallel genotyping of pooled DNA

Minor histocompatibility (H) antigens are the molecular targets of allo-immunity responsible both for the development of antitumor effects and for graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, despite their potential clinical use, our knowledge of human minor H antigens is largely limited by the lack of efficient methods of their characterization. Here we report a robust and efficient method of minor H gene discovery that combines whole genome association scans (WGASs) with cytotoxic T-lymphocyte (CTL) assays, in which the genetic loci of minor H genes recognized by the CTL clones are precisely identified using pooled-DNA analysis of immortalized lymphoblastoid cell lines with/without susceptibility to those CTLs. Using this method, we have successfully mapped 2 loci: one previously characterized (HMSD encoding ACC-6), and one novel. The novel minor H antigen encoded by BCL2A1 was identified within a 26 kb linkage disequilibrium block on chromosome 15q25, which had been directly mapped by WGAS. The pool size required to identify these regions was no more than 100 individuals. Thus, once CTL clones are generated, this method should substantially facilitate discovery of minor H antigens applicable to targeted allo-immune therapies and also contribute to our understanding of human allo-immunity.