Differential signalling mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNFalpha

To gain a clearer insight into the mechanisms of skeletal muscle cell growth, differentiation and maintenance, we have developed a primary adult human skeletal muscle cell model. Cells were cultured from biopsies of rectus muscle from the anterior abdominal wall of patients undergoing elective surgery. Under differentiating conditions, all cultures formed myotubes, irrespective of initial myoblast number. Stimulation with both IGF-I and tumour necrosis factor alpha (TNFalpha) increased cellular proliferation but while IGF-I subsequently increased myoblast differentiation, via both hyperplasia and hypertrophy, TNFalpha inhibited the initiation of differentiation, but did not induce apoptosis. Addition of IGF-I stimulated both the MAP kinase and the phosphatidylinositide 3-kinase (PI 3-kinase) signalling pathways while treatment with TNFalpha preferentially led to MAP kinase activation although with a very different profile of activation compared to IGF-I. Data using the MEK inhibitor UO126 showed MAP kinase activity is not only needed for cellular proliferation but is also necessary for both the initiation and the progression of primary human myoblast differentiation. The PI 3-kinase pathway is also involved in differentiation, but activation of this pathway could not relieve inhibition of differentiation by TNFalpha or UO126. Our results show that the controlled temporal and amplitude of activation of multiple signalling pathways is needed for successful myoblast differentiation.     

The binding of complement by complexes formed between a rabbit antibody and oligosaccharides of increasing size.

Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.     

Influence of circadian time and age on glomerular angiotensin II receptors in normotensive Sprague-Dawley and transgenic hypertensive TGR(mREN2)27 rats

In male heterozygous transgenic hypertensive rats, TGR(mREN2)27 (TGR), exhibiting an inverse blood pressure profile and in normotensive Sprague-Dawley (SPRD) controls, the density and affinity of angiotensin II receptors were determined at six circadian times in glomeruli of animals 11 weeks old kept under light-dark 12h:12 (LD 12:12) conditions. Angiotensin II receptors were also studied in rats 18-20 weeks old of both strains at 2h after light onset. As a measure of renal excretory functions, diuresis, creatinine, and protein excretion were monitored using metabolic cages. The expression of angiotensin II receptor mRNA was determined in renal arteries 2h-4h after light onset. The following results were obtained: [1] Renal excretory functions showed significant daily variation, with higher excretion rates in the dark span in both TGR and SPRD rats. [2] No circadian phase dependency was found in the glomerular angiotensin II receptors in both rat strains. However, receptor density was significantly lower in TGR than in SPRD rats. In both strains, receptor number increased with aging. [3] In renal arteries, the angiotensin II receptor mRNA of the main receptor subtype AT_1A was neither strain nor age dependent, AT_1B- and AT₂-receptor mRNAs were significantly lower in TGR than SPRD rats. In conclusion, the results demonstrate that the overactive renin-angiotensin system in TGR rats led to a down-regulation of glomerular angiotensin II receptors that was not accompanied by a down-regulation of the mRNA of the dominant AT_1A- receptor subtype. Circadian short-term variations in blood pressure in both TGR and SPRD rats are not reflected by daily variation in angiotensin II receptor density of renal glomeruli or by variation in receptor expression in renal vascular tissue. (Chronobiology International, 18(3), 447-459, 2001)     

Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.     

Tracking and quantitation of fluorescent HIV during cell-to-cell transmission

The green fluorescent protein (GFP) is a powerful genetic marking tool that has enabled virologists to monitor and track viral proteins during HIV infection. Expression-optimized Gag-GFP constructs have been used to study virus-like particle (VLP) assembly and localization in cell types that are easily transfected. The development of HIV-1 variants carrying GFP within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells following virological synapse formation. HIV Gag-iGFP, a viral clone that contains GFP inserted between the matrix (MA) and capsid (CA) domains of Gag, is the first replication competent molecular clone that generates fluorescent infectious particles. Here, we discuss some methods that exploit HIV Gag-iGFP to quantify cell-to-cell transmission of virus by flow cytometry and to track the proteins during assembly and transmission using live-cell imaging.     

Sensitivity of Heart Rate and Blood Pressure to Spontaneous Activity in Transgenic Rats

Spontaneous changes in heart rate (HR), activity and systolic (SBP) and diastolic (DBP) blood pressure have been measured in 3 groups of 7 transgenic [TGR(mRen-2)27] rats for 4 weeks, starting at 12 weeks of age, and living on a 12:12 L:D schedule (light on at 07:00 h). Group TG-ENA was given enalapril, an angiotensin-converting enzyme inhibitor, in its drinking water; group TG-AMLO was given the calcium-channel blocker, amlodipine, by the same route; and group TG-VEH had no addition to its drinking water and so acted as a control. The sensitivity of the cardiovascular variables (CV's) to spontaneous activity was assessed throughout the study period by measuring the gradient of [CV / activity]. For the control (TG-VEH) group, mean HR was highest during the dark phase, at which time the sensitivity to spontaneous activity was least. By contrast, the circadian rhythms of SBP and DBP were inverted, peaking in the light (resting) phase, and there was no reliable difference between the light and dark phases with regard to the sensitivity of SBP or DBP to the effects of spontaneous activity. Enalapril reduced SBP and DBP, but did not alter their phase inversion with respect to HR. However, in SBP and DBP, as well as HR, sensitivities to spontaneous activity were now greater in the light phase. Amlodipine also reduced SBP and DBP and, in addition, greatly reduced the amplitude of their circadian rhythms. With this treatment also, sensitivity to spontaneous activity was greatest in the light phase for HR, SBP and DBP. A simple explanation of these results is that, in the absence of treatment, transgenic rats of this age have DBP and, particularly, SBP values that are too high in the light (resting) phase to permit much further rise due to spontaneous activity, and that this "ceiling effect" no longer holds if SBP and DBP have been reduced pharmacologically.     

River macrophyte indices: not the Holy Grail!

1. Recent studies have demonstrated that there is generally no unambiguous relationship between plant species composition and specific environmental conditions in rivers. Nevertheless, indices of environmental pressures based on macrophytes are flourishing, because of the requirements of the Water Framework Directive (WFD). 2. We first reviewed nine such indices against 13 criteria for bioindicators. Then, using data from France and England, we tested whether the IBMR (Macrophyte Biological Index for Rivers) and LEAFPACS (predictions and classification system for macrophytes) methods could reliably indicate nutrient and hydromorphological pressures. Finally, we used an improved bootstrapping method to estimate accuracy. 3. Currently, most indices lack ecological meaning for a variety of reasons, including partial sampling (backwaters are excluded); reliance on list of taxa (there are identification difficulties) rather than structure and functions; correlation rather than causation; application within a limited biogeographical area; reliance on ‘expert’ judgement; high precision but poor accuracy; poorly defined reference conditions; lack of independent tests; and an inability to discriminate reliably between the target pressures of interest from confounding background variables. 4. IBMR was a far better indicator of pH (or HCO₃‐pCO₂) than it was of soluble reactive phosphorus, SRP (or SRP‐NH₄). While there was a highly significant correlation between IBMR and SRP after removing the effect of pH, the relationship was weak (r² = 0.08, n = 215, P < 0.001). 5. LEAFPACS is a multi‐metric method summing up five individual indices, each compliant with the WFD. Its individual metrics were not better correlated with nutrient and hydromorphological pressures (with r² < 0.1, n = 62, P < 0.05) than was the IBMR. The meaning of the overall metric is questionable. 6. There are problems in determining the precision of the indices, owing to uncertainties in recording, but they are less than the uncertainties in determining accuracy (because species optima and tolerances are sometimes poorly known). 7. Reliable information is needed to improve the state of our rivers. Macrophyte indices are able to detect statistically significant pressures from a large population of sites but cannot be applied at specific sites, as required by the WFD, owing to large uncertainties and low explanatory power. Typically, more than 90% of the variability in macrophyte indices is attributed to factors other than human pressure. The WFD would be better served by a simpler, holistic approach based on our current mechanistic understanding of river processes. These findings are likely to apply also to other taxonomic groups (macroinvertebrates, diatoms, fish) used in the assessment of purported ecological quality and to palaeolimnological measures of reference status.     

Detection of Plant-Modulated Alterations in Antifungal Gene Expression in Pseudomonas fluorescens CHA0 on Roots by Flow Cytometry

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.     

Nano-biophotonics: new tools for chemical nano-analytics

The nondestructive chemical analysis of biological processes in the crowded intracellular environment, at cellular membranes, and between cells with a spatial resolution well beyond the diffraction limit is made possible through Nano-Biophotonics. A number of sophisticated schemes employing nanoparticles, nano-apertures, or shaping of the probe volume in the far field have significantly extended our knowledge about lipid rafts, macromolecular complexes, such as chromatin, vesicles, and cellular organelles, and their interactions and trafficking within the cell. Here, I review some of the most recent developments in Nano-Biophotonics that already are or soon will become relevant to the analysis of intracellular processes. The pros and cons of the various techniques will be discussed and an outlook of their prospects for the near future will be provided.