An encore for ribosome biogenesis in the control of cell proliferation

Although the importance of cell growth for cell-cycle progression has been recognized for thirty years, the molecular basis of this relationship is poorly understood. However, researchers have begun to tease apart these two processes in model systems. This commentary focuses on one potential mechanism by which ribosome biogenesis antagonizes cell-cycle progression until the cell has grown to an adequate size.     

High-performance liquid chromatography with on-line post-column immunoreaction detection of digoxin and its metabolites based on fluorescence energy transfer in the far-red spectral region

The combination of immunoassays with separation techniques such as chromatography can result in enhanced selectivity and sensitivity. This paper describes an on-line chromatography with immunochemical post-column fluorescence energy transfer detection for digoxin and its metabolites. R-phycoerythrin (PE) was used as the donor and an indodicarbocyanine dye (Cy5) as the acceptor label. These labels allow the detection in the far-red spectral region, which is more selective for biological samples. Hence, digoxin was labeled with PE using the activated digoxigenin-NHS-ester and monoclonal anti-digoxin antibody was labeled with Cy5. Digoxin and its metabolites was injected into the HPLC system followed by post-column injection of R-phycoerythrin labeled digoxin and by Cy5 labeled anti-digoxin antibody. Incubation time was provided using an open tubular reactor coil at room temperature. The detection was performed by measurement of the sensitized emission of Cy5 at 670 nm due to fluorescence energy transfer from PE labeled with digoxin. The system was optimized with regard to the concentrations of the used post-column reagents as well as incubation time and temperature. The dynamic range of digoxin spiked in 0.01 M phosphate buffer (pH 7.4) was 0.05 to 10 ng/ml with a correlation coefficient of 0.989. The limit of detection was 33 pg/ml. The precision of two controls, 0.4 and 4 ng/ml, was found to be 2.2 and 8.7% RSD, respectively, accuracy was 10.7 and 20.3% (n=6 in each case).     

Gonadal steroid preservation of central synaptic input to hamster facial motoneurons following peripheral axotomy

In recent work, we have demonstrated that testosterone propionate accelerates recovery from facial nerve injury in the adult male hamster. Central synaptic stripping following peripheral motor neuron damage is a well-established component of the injury response. Gonadal steroids regulate synaptogenesis in the normal nervous system. In this study, we tested the hypothesis that testosterone propionate administration at the time of facial nerve transection alters the synaptic connectivity of injured facial motoneurons. Adult hamsters were subjected to right facial nerve transection at the level of the stylomastoid foramen. Half the animals received subcutaneous implants of testosterone propionate; the other half were sham implanted. At 5 days postoperative, the animals were killed by intracardiac perfusion-fixation, and the control and axotomized facial nuclear groups from the brainstems of nonhormone- and testosterone propionate-treated animals processed for routine transmission electron microscopy. Quantiative analysis of the synaptic ratio (percent somal membrane covered by synaptic profiles) and the average length of axosomatic synapses was accomplished. The results indicate that axotomy alone resulted in an 81% reduction in the synaptic ratio and a 26% decrease in the average synaptic length of axosomatic synapses. Exposure to testosterone propionate from the time of facial nerve transection resulted in only a 48% reduction in the synaptic ratio and a 16% decrease in the average synaptic length of axosomatic synapses following injury. Thus, testosterone propionate significantly attenuated the amount of synaptic stripping that occurred at 5 days postoperative and the decrease in average length of the remaining synapses as well. It is concluded that gonadal steroids modulate central synaptic plasticity following peripheral nerve injury. The results are discussed in light of our recent findings of steroidal effects on the central astrocyctic response to facial nerve injury as well.     

Homology Modelling of a Newly Discovered Thioredoxin Protein and Analysis of the Force Field and Electrostatic Properties

Thioredoxin is a small protein (Mr approximately 12,000) found in all living cells from archaebacteria to humans. The active site is highly conserved and has two redox-active cysteine residues in the sequence: -Trp-Cys-Gly-Pro-Cys-. Besides the function of the reduced form as a powerful protein disulfide oxidoreductase, thioredoxin is known to regulate and activate different target enzymes, i.e. ribonucleotide reductase and the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. Despite the high degree of homology between thioredoxin proteins from different species, there exists a strong variation in the capability of activating target enzymes. This is yet unexplainable, since there still exists no model of a thioredoxin/receptor complex.On the basis of the recently determined amino acid sequence of the thioredoxin Trx2 from rat mitochondria, which is known to be highly efficient in activating mitochondrial 2-oxoacid dehydrogenase multienzyme complexes, we construct the 3-D structure of this protein by homology modelling methods, using the X-ray structures of thioredoxin from E. coli and human as background information. We analyze the differences in the electrostatic properties of the different protein structures and show, that despite the observed homology between the primary sequences, the dipole moment of the protein structures shows significant variations, which might lead to deviations with respect to the binding to the target protein. Using the AMBER 4.0 program package we further investigate and compare the force field energies of the different thioredoxin structures.Electronic Supplementary Material available.     

Combined genetic polymorphism and risk for development of lung cancer

Susceptibility to lung cancer has been shown to be modulated by host specific factors. Inheritance of different polymorphic cytochrome P450s (CYPs) and the glutathione S-transferases (GSTs) which affect metabolism of environmental toxicants may play a key role in individual susceptibility. Although individual polymorphic genes have been reported to be associated with development of lung cancer, little is known about the combined effects of several genes in carcinogenesis. From our study of 54 lung cancer patients and 50 matched controls, we observed that a combination of several versions of ‘unfavorable' metabolizing genes (CYP2D6, CYP2E1, GSTM1 and GSTT1) is strongly associated with lung cancer. The relative risk for the different combinations of these genotypes ranged between 1.3 and 14, with higher risk involving the activating genes. The duration and intensity of heavy smoking (expressed in pack-years) are the most important determinant for the development of lung cancer. For example, the estimated risk for development of lung cancer associated with smoking >30 pack-years is represented by an odds ratio=6.65; 95% CL=2.3–19.9 irrespective of an individual's genotype, whereas for smoking between >30 and <50 pack-years, odds ratio=4.5; 95% CL=1.37–15; and for smoking >50 pack-years, odds ratio=30; 95% CL=5.7–114. On the other hand, smoking of less than 30 pack-years is associated with an increased risk in the presence of the polymorphic genes (odds ratio=2.5; 95% CL=0.32–54). The results of our study indicate that the inheritance of multiple ‘unfavorable' genotypes, especially activating genes, is a crucial predisposing factor for the development of lung cancer from cigarette smoking. In addition, the genes may cause moderate smokers who would normally outlive the deleterious effects of smoking to develop lung cancer. The information can therefore be used to target individuals for prevention of health problems.     

Elevated frequencies of hypoxanthine phosphoribosyltransferase lymphocyte mutants are detected in Russian liquidators 6 to 10 years after exposure to radiation from the Chernobyl nuclear power plant accident

This study was conducted to determine whether the frequency of hypoxanthine phosphoribosyltransferase (HPRT) deficient lymphocyte mutants would detect an effect of radiation exposure in a population of Russians who were exposed to low levels of radiation while working in 1986 and 1987 as liquidators cleaning up after the Chernobyl nuclear power reactor accident. The HPRT lymphocyte cloning assay was performed on peripheral blood lymphocytes collected between 1992 and 1996 from 142 liquidators and 66 Russian controls, and between 1989 and 1993 from 231 American controls. Russian and American controls were not significantly different for either cloning efficiency or mutant frequency (MF); inclusion of both sets of controls in the analysis increased the ability to detect a Chernobyl exposure effect in the liquidators. After adjusting for age and smoking, the results revealed no significant difference in cloning efficiency of Chernobyl liquidators relative to Russian controls but a significant, 24% increase in liquidator HPRT mutant frequency over Russian controls (90% confidence interval was 7% to 45% increase). The analytical method also accounted for differences in precision of the individual estimates of log CE and log MF and accommodated for outliers. The increase in HPRT mutant frequency of liquidators is an attribute of the exposed population as a whole rather than of individuals. These results demonstrate that, under appropriate circumstances, the HPRT specific locus mutation assay of peripheral blood lymphocytes can be used to detect a semi-acute, low dose radiation exposure of a population, even 6 to 10 years after the exposure.     

Genomic analysis of adaptive differentiation in Drosophila melanogaster

Drosophila melanogaster shows clinal variation along latitudinal transects on multiple continents for several phenotypes, allozyme variants, sequence variants, and chromosome inversions. Previous investigation suggests that many such clines are due to spatially varying selection rather than demographic history, but the genomic extent of such selection is unknown. To map differentiation throughout the genome, we hybridized DNA from temperate and subtropical populations to Affymetrix tiling arrays. The dense genomic sampling of variants and low level of linkage disequilibrium in D. melanogaster enabled identification of many small, differentiated regions. Many regions are differentiated in parallel in the United States and Australia, strongly supporting the idea that they are influenced by spatially varying selection. Genomic differentiation is distributed nonrandomly with respect to gene function, even in regions differentiated on only one continent, providing further evidence for the role of selection. These data provide candidate genes for phenotypes known to vary clinally and implicate interesting new processes in genotype-by-environment interactions, including chorion proteins, proteins regulating meiotic recombination and segregation, gustatory and olfactory receptors, and proteins affecting synaptic function and behavior. This portrait of differentiation provides a genomic perspective on adaptation and the maintenance of variation through spatially varying selection.