The MyoD family of transcription factors and skeletal myogenesis

Gene targeting has allowed the dissection of complex biological processes at the genetic level. Our understanding of the nuances of skeletal muscle development has been greatly increased by the analysis of mice carrying targeted null mutations in the Myf-5, MyoD and myogenin genes, encoding members of the myogenic regulatory factor (MRF) family. These experiments have elucidated the hierarchical relationships existing between the MRFs, and established that functional redundancy is a feature of the MRF regulatory network. Either MyoD or Myf-5 is sufficient for the formation or survival of skeletal myoblasts. Myogenin acts later in development and plays an essential in vivo role in the terminal differentiation of myotubes.     

Expression of a pepT homologue from Bacillus subtilis

Abstract We isolated pepT from Bacillus subtilis , a gene with homology to various tripeptidases from different bacterial sources, pepT is preceded by genes encoding a two component regulatory system. Its expression is activated during stationary phase. In minimal medium this activation is boosted in the presence of phosphate. The response regulator is preceded by putative promoter consensus sequences recognized by the stationary phase specific sigma factors σ ^H, σ ^F, and σ ^G. This is in accordance with the initiation of expression at the beginning of stationary phase. Inactivation of pepT causes no obvious phenotype.     

Subordinates explore but dominants profit: resource competition in high Arctic barnacle goose flocks

Social dominance plays an important role in assessing and obtaining access to patchy or scarce food sources in group-foraging herbivores. We investigated the foraging strategies of individuals with respect to their social position in the group in a flock of nonbreeding, moulting barnacle geese, Branta leucopsis, on high Arctic Spitsbergen. We first determined the dominance rank of individually marked birds. The dominance of an individual was best described by its age and its sex-specific body mass. Mating status explained the large variation in dominance among younger birds, as unpaired yearlings ranked lowest. In an artificially created, competitive situation, subordinate individuals occupied explorative front positions in the flock and were the first to find sites with experimentally enriched vegetation. Nevertheless, they were displaced quickly from these favourable sites by more dominant geese which were able to monopolize them. The enhanced sites were subsequently visited preferentially by individuals that succeeded in feeding there when the exclosures were first opened. Data on walking speed of foraging individuals and nearest-neighbour distances in the group suggest that subordinates try to compensate for a lower energy intake by exploring and by lengthening the foraging bout. Observations of our focal birds during the following breeding season revealed that females that returned to the study area were significantly more dominant in the previous year than those not seen in the area again. Copyright 2001 The Association for the Study of Animal Behaviour.     

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (v _max=24.4 pmol/(mg min), K _m=64.4M) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.     

Multichannel apparatus for parallel monitoring of light scattering in Dictyostelium discoideum cell suspensions

Suspensions of Dictyostelium discoideum amoebae display free-running light scattering oscillations at the onset of development. We describe a device to monitor these oscillations in several samples in parallel. The apparatus consists of a thermostated cuvette holder where up to eight cuvettes containing cell suspension are inserted. Cells are aerated and kept in suspension via an airlift. Infrared light emitted from a five-diode array passes through the suspension and is detected by an array of five light detecting diodes. The resulting signal is digitized and recorded with a sampling rate of two measuring points/second. The parallel analysis approach allows determination of the effects of adding of agents or of variations in the external conditions in the same batch of amoebae at the same developmental time point. This represents an advantage over the conventional single cuvette approach, as oscillation characteristics themselves are developmentally regulated. Moreover, as the new experimental setup enables simultaneous analyses of up to eight samples, the behavior of wild-type and several mutant strains can be compared under identical experimental conditions.     

Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells

The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells. The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC. Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells. Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC. Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA. This was also true for glutathione-depleted cells. The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.     

Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling

In vertebrates, PDGFA and its receptor, PDGFRalpha, are expressed in the early embryo. Impairing their function causes an array of developmental defects, but the underlying target processes that are directly controlled by these factors are not well known. We show that in the Xenopus gastrula, PDGFA/PDGFRalpha signaling is required for the directional migration of mesodermal cells on the extracellular matrix of the blastocoel roof. Blocking PDGFRalpha function in the mesoderm does not inhibit migration per se, but results in movement that is randomized and no longer directed towards the animal pole. Likewise, compromising PDGFA function in the blastocoel roof substratum abolishes directionality of movement. Overexpression of wild-type PDGFA, or inhibition of PDGFA both lead to randomized migration, disorientation of polarized mesodermal cells, decreased movement towards the animal pole, and reduced head formation and axis elongation. This is consistent with an instructive role for PDGFA in the guidance of mesoderm migration.     

Transrectal Doppler sonography of uterine and umbilical blood flow during pregnancy in mares

Transrectal color Doppler sonography was used to investigate uterine and umbilical blood flow during pregnancy (duration, 46-48 weeks) in four mares. The resistance index (RI) and blood flow volume (VOL) of the uterine arteries ipsilateral and contralateral to the conceptus, and the presence of an early diastolic notch in the Doppler wave, were evaluated every 4 week throughout pregnancy. Fetal blood flow was calculated semiquantitatively every 2 week (from 20 to 40 weeks), using the RI of the umbilical arteries. During the entire period of investigation, there were no significant individual variations in uterine RI and VOL nor differences between the two uterine arteries. Mean RI decreased by more than half during pregnancy from 0.89 +/- 0.01 to 0.39 +/- 0.03, and mean VOL increased almost 400-fold from 69 +/- 37 to 27,467 +/- 8851 ml/min. There were relationships (P<0.0001) between week of pregnancy (x) and RI as well as VOL. These were described by the equations RI=0.938-0.150 ln(x) and VOL (ml/min)=7.621x(2.157). Log transformed total estrogen (TE) were related to RI (r=-0.879; P<0.05) as well as to VOL (r=0.888; P<0.05). The notch in the Doppler wave of the uterine artery disappeared between 18 and 26 weeks. There was a correlation (P<0.0001) between week of gestation (x) and RI values of the umbilical arteries; this was described by the equation RI=1.763-0.071x+0.001x2. Further studies are needed to determine whether transrectal color Doppler sonography could be used to identify mares at risk of abortion.     

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.     

Seasonal variation in the microbial contamination of game carcasses in an Austrian hunting area

In 2003, 50 game carcasses (ungulates) originating from one Austrian hunting ground were subject to visual examination for (fecal) contamination of the body cavities and microbiological testing of the body cavities in order to assess variations in microbial surface contamination in the season June–August compared to October–December. No carcass tested positive for the bacterial pathogens Salmonella or Listeria. Bacterial surface counts in October–December (median values: total aerobic count: 4.12 log₁₀ colony-forming-units (cfu)/cm²; Enterobacteriaceae: 2.48 log₁₀ cfu/cm²) were significantly lower than those in June–August (median values: total aerobic count: 5.65 log₁₀ cfu/cm²; Enterobacteriaceae: 3.45 log₁₀ cfu/cm²). The cooling regime (0.4 °C, 62% relative humidity) allowed no microbial growth for 96 h but was associated with weight loss of the carcasses. All carcasses had undergone a precooling phase of 8–12 h, with temperatures of 17.8±1.2 °C in the season June–August and 9.8±1.2 °C in October–December. This temperature difference was identified as the most probable effector for the observed seasonal variation. The results demonstrate the need for a continuous cool chain after evisceration of game carcasses.