Deciphering the molecular machinery of stem cells: a look at the neoblast gene expression profile

Background  

Mammalian stem cells are difficult to access experimentally; model systems that can regenerate offer an alternative way to characterize stem cell related genes. Planarian regeneration depends on adult pluripotent stem cells - the neoblasts. These cells can be selectively destroyed using X-rays, enabling comparison of organisms lacking stem cells with wild-type worms.     

Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae

Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h^–1on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae.     

DNA methylation by tert-butyl hydroperoxide-iron (II): a role for the transition metal ion in the production of DNA base adducts.

Metabolic degradation of both endogenous and exogenous peroxides is associated with the etiology of several diseases including cancer. Tert-butyl hydroperoxide (TBHP) has been widely employed as a model compound to study the cytotoxicity and promoting effects of organic peroxides. Recently, we reported that incubations of TBHP with iron (II) and calf thymus DNA led to generation of high yields of methyl radicals and to DNA methylation. Interestingly, DNA was methylated to products expected from both free radical and ionic mechanisms such as 8-methylguanine (C8-MeGua) and 7-methylguanine (N7-MeGua), respectively. To elucidate the mechanisms by which methyl radicals can produce different types of DNA adducts, we examined the effects of transition metal ions (iron (II), iron (III) and copper (I)) and metal ion chelators (ethylenediamine-N,N,N",N"-tetraacetate (EDTA) and desferal) on the nature and the yields of the DNA adducts produced during TBHP decomposition. The results led us to propose that a direct methyl radical attack on DNA guanine residues produces C8-MeGua whereas N7-MeGua and 3-methyladenine (N3-MeAde) are likely to be produced by attack of nucleophilic DNA centers on methyl radical generated in situ by the assistance of transition metal ions bound to DNA.     

The acetyl xylan esterase II gene from Penicillium purpurogenum is differentially expressed in several carbon sources, and tightly regulated by pH

The expression of the acetyl xylan esterase II (axeII) gene from Penicillium purpurogenum is repressed by glucose and induced by xylan, as well as to a small degree by xylose and xylitol. This gene is expressed at neutral pH, but not under alkaline or acidic conditions, in agreement with previous findings for other xylanolytic genes of this organism. This is the first report showing pH regulation of an axe gene.     

A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM)

In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a reduction of the amount of drug needed for testing, which is crucial for screening new molecules, when many different toxicological tests have to be carried out. The microassay is therefore a useful and reproducible tool for screening compounds (chemicals, drugs and xenobiotics) for potential haematotoxicity directly on human myeloid progenitors, and could contribute significantly to reducing the use of animals in toxicity testing.     

Frontal cortex: goal-relatedness and the cortical motor system

A recent study has shown that prolonged electrical stimulation of the ventral premotor cortex can evoke complex defensive movements. Moreover, neurons in this region show activity correlated with the vigour of an induced defensive reaction. These results support the idea that this cortical region encodes goal-related actions.