Analysis of the expression of chicken and rat gene products in myoblast x myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.     

The ant Lasius niger shows no relationship between task efficiency and body size variation among workers

In ants, workers of different sizes may perform various tasks, even in so-called monomorphic species with relatively low body size variation. However, it is unclear if the body size diversity of monomorphic workers correlates with task efficiency, especially in stressful contingencies. Here we tested if the body size variation of workers corresponds with its efficiency in transferring pupae. Transferring brood is a pre-set behavioral response to stress, e.g. suboptimal temperature. Here we applied a laboratory experiment simulating nest damage. The study was performed on the common garden ant (Lasius niger (Linnaeus, 1758)) – a species with no distinct worker subcastes. The efficiency of workers was measured as the latency of transferring pupae from a lit part of the experimental colony to a darkened part, while the body size diversity was expressed as the within-colony coefficient of variation in head width. We did not find any significant correlation between efficiency and body size variation. Summarizing the existing studies and the present results, we propose the hypothesis that the body size diversity of L. niger may have implications for workers’ division of labor but not for their task efficiency in a stressful contingency.     

Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [³H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G₀ stage of the cell cycle.     

Analysis of the expression of chicken and rat gene products in myoblast × myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.     

The use of supramolecular structures as protein ligands

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.     

Population and community-level compositional patterns shape the realized niche of the rare arctic-alpine species Carex lachenalii Schkuhr

The ability for vegetative growth and development of generative organs often reflects an adaptation to the environment and may be a suitable proxy for understanding population dynamics of rare relict species. An example of such a plant is Carex lachenalii Schkuhr, an arctic-alpine species, in the temperate zone of Europe only occurring in isolated localities of high-elevation mountain ranges. We aimed to assess whether there were relationships between flower production and clonal growth of C. lachenalii, both at the tuft and plot level, and how co-occurring vegetation could modify this relationship. In the study we focused on population-level traits of C. lachenalii, vegetation traits and components of functional diversity. At the tuft level we found that the proportion of flowering ramets of C. lachenalii decreased with increasing diameter of the tuft. At the plot level, in snowbed vegetation C. lachenalii produced more flowering ramets. We suggest this is due to higher environmental stress, expressed by high importance of habitat filtering (low functional dispersion) in shaping species composition of co-occurring vegetation. In granite grasslands and milder environment (expressed by higher functional dispersion), C. lachenalii produced more vegetative ramets, which we suggest is a result of a more competitive environment. While in snowbeds investment in flowering ramets could promote successful persistence of C. lachenalii, survival of subpopulations occurring in the highly competitive conditions of granite grasslands may be uncertain due to potentially weak adaptation to competition with graminoids and dwarf shrubs.     

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.     

Modulation of ERK1/2 activity is crucial for sphingosine-induced death of glioma C6 cells

In this study the contribution of the ERK1/2 pathway to sphingosine-induced death and morphological changes of the actin cytoskeleton in glioma C6 cells was investigated. Surprisingly, the level of ERK1/2 phosphorylation does not change after incubation of cells with sphingosine. Despite this, sphingosine induces rounding and detachment of cells without formation of apoptotic bodies. To shed light on this process, a specific inhibitor of ERK1/2 phosphorylation, U0126, was used. Cells incubated simultaneously with sphingosine and U0126 not only detached, but also exhibited formation of apoptotic-like blebs. These data suggest that during sphingosine-induced glioma C6 cell death apoptotic blebbing is dependent on ERK1/2 signalling and occurs only when ERK1/2 activity is decreased or abolished.