Joint participation of mitochondria and sarcoplasmic reticulum in the formation of tubular aggregates in gastrocnemius muscle of CK-/- mice

Tubular aggregates are specific subcellular structures that appear in skeletal muscle fibres under different pathological conditions. The origin of the tubular aggregates is generally ascribed to proliferating membranes of sarcoplasmic reticulum. There are, however, histochemical indications for the presence of mitochondrial enzymes in tubular aggregates suggesting contribution of mitochondria to the genesis of tubular aggregates. In this study we used an immunocytochemical detection technique to assess participation of mitochondria and of sarcoplasmic reticulum in derivation of tubular aggregates. The fast skeletal muscle fibres (m. gastrocnemius) of mice bearing the double invalidation for both the mitochondrial and the cytosolic isoforms of creatine kinase (CK), an enzyme involved in energetics of muscle cells, were employed as a model muscle with tubular aggregates (Steeghs et al., Cell 89, 93-103, 1997). Immunogold labelling of the bc1 complex, a specific integral protein of the inner mitochondrial membrane, provided strong signals in both the mitochondria and tubular aggregates but not in other ultrastructural components of muscle fibres. A similar strong immunogold signal was obtained when labelling for SERCA1, a specific enzyme of the sarcoplasmic reticulum membrane, in regions of typical occurrence of the sarcoplasmic reticulum and in tubular aggregates. In double labelling experiments, we found simultaneous labelling of tubular aggregates with both the bc1 and SERCA1 antibodies. It is concluded, that in CK-/- mouse both the inner mitochondrial membrane and the membrane of the sarcoplasmic reticulum participate in the formation of tubular aggregates.     

Chromosomal R-banding with a monoclonal antidouble-stranded DNA antibody

Summary A monoclonal anti-DNA antibody (HB2) specific for poly dG-poly dC nucleotides was used to stain metaphasic lymphocyte or amniotic cell human chromosomes. HB2 fixation was revealed using either a peroxidase-or a rhodaminelabeled anti-mouse immunoglobulin antiserum. The staining pattern of the chromosomes was dependent on the HB2 concentration: R-banding could be observed at high antibody dilution. Previous trypsinization of metaphasic preparations demonstrated a precise and reproducible typical R-banding independent of the HB2 concentration. This technique appears to be an interesting alternative to other R-banding procedures. The specificity of the antibody allows a better understanding of the biochemical mechanism of R-banding.     

Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate.

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.     

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.     

Environmental impact of neolithic and bronze age farming in the eastern Pyrenees forelands, based on multidisciplinary investigations at La Caune de Bélesta (Bélesta Cave), near Perpignan, France

Results of multidisciplinary studies, involving anthracology, archaeology and geoarchaeology, that have been carried out on Neolithic to Bronze Age deposits from Bélesta Cave, eastern Pyrenees, are reported. These show that the type of human activity, i.e. pastoralism alone or a more diversified farming economy, and continuity/discontinuity of occupation are the main factors that determine the structure and evolution of the Holocene vegetation in the region. Neolithic pastoral activities were not continuous and so did not have an enduring influence on the natural environment. The more continuous and diversified exploitation associated with Bronze Age cultures was responsible for the development and maintenance of the low garrigues.     

Suppression of the late stages of mitogen-induced human B cell differentiation by FC gamma receptors (FC gamma R) released from polymorphonuclear neutrophils

During short incubation in serum-free medium, polymorphonuclear neutrophils (PMN) release soluble material that can be characterized as receptors for Fc IgG (Fc gamma R) on the following evidence: it agglutinates erythrocyte-IgG antibody (EAG) complexes, it prevents the binding of EAG to EAG-rosette-forming cells, and it binds to EAG-rosette-forming cells after modulation of their Fc gamma R, allowing the formation of 'passive' rosettes. These Fc gamma R were isolated by affinity chromatography on sepharose 4B-IgG. This material was shown to interfere with the differentiation of peripheral blood B cells into Ig-secreting cells in cultures stimulated by pokeweed (PWM) or Nocardia opaca (NOC) extracts. The number of Ig-secreting cells determined by a reverse hemolytic plaque assay using protein A-coated sheep erythrocytes was decreased by addition of Fc gamma R over a wide range of dilutions. The number of Ig-containing cells was diminished in PWM-stimulated cultures, but not in cultures stimulated with NOC. Fc gamma R did not affect cell viability, nor did they interfere with plaque-forming cell assay. Fc gamma R was not suppressive when added before the 3rd day or after the 6th day of culture. The suppressor factor was shown to remain associated with Fc gamma R after elution at acidic pH; it was removed by absorption on Sepharose 4B-IgG, but not on pepsin-digested F(ab')2 fragments. The suppressive factor as well as the capacity to restore EAG rosette formation by modulated lymphocytes were destroyed by heating (56 degrees C, 30 min) or by freezing and thawing. Properties of Fc gamma R released from PMN in this system are similar to those of Fc gamma R released from unstimulated human peripheral blood lymphocytes (PBL) and to those of mouse Ig-binding factor produced by alloactivated T cells or T cell lines.     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Higher-level classification of the Archaea: evolution of methanogenesis and methanogens

We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO(2)) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO(2), formate, acetate, methylated C-1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon.