The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.     

Analysis ofl-alanine:2-oxoglutarate aminotransferase isozymes in maize

Isozyme analysis ofl-alanine:2-oxoglutarate aminotransferase (ALT) in maize indicates that there are three genes encoding this enzyme activity. Two of the gene products interact with each other to form heterodimers, while the third gene product does not interact with the other two. Another isozyme that appears after gel electrophoresis and ALT staining is shown to be glutamate dehydrogenase-1. Anaerobic treatment does not result in increased ALT levels, indicating that the previously reported increase in alanine levels caused by this treatment may be due to increases in the level of pyruvate, a substrate of ALT.D. A. Russell was partially supported by a graduate student fellowship from the Division of Biology and Biomedical Sciences, Washington University. V. M. Peschke was partially supported by a postdoctoral fellowship from Monsanto. This research was supported by NIH Grant R01 GM34740.     

Two-step purification of full-length nerve growth factor receptor and maintenance of receptor-specific binding activity

A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.     

NMR Studies of Pi-Containing Extracellular and Cytoplasmic Compartments in Brain

Abstract: The inorganic phosphate (P_i) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of P_i. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated P_i spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of P_i buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the P_i peaks arising from the three compartments with different pH values if each peak made up 10–35% of total P_i area. In vivo, we identified the plasma compartment by intraarterial infusion of P_i. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that P_i compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH_i. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger P_i peaks represented intracellular spaces.     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Computational sequence analysis of matrix metalloproteinases

Matrix metalloproteinases (MMP) play a cardinal role in the breakdown of extracellular matrix involved in a variety of biological and pathological processes. Research on MMPs has classified and characterized these enzymes according to their matrix substrate specificity, gene and protein domain structure, and regulation of activity and expression. However, the discovery of new MMPs has introduced a need for a more comprehensive and systematic method of classification and quantitative comparison of known and newly discovered members. This study compiles a sequence alignment, constructs a dendrogram, and calculates physical data and homology percentage assignments in order to obtain further insight into MMP structure-function relationships. Thorough analysis of MMP primary sequence domains, physical data patterns, and statistical analysis of sequence homology yields higher resolution in the similarities and differences that group MMP members.     

Higher level relationships of Apiales (Apiaceae and Araliaceae) based on phylogenetic analysis of rbcL sequences

The two families of the order Apiales (Apiaceae and Araliaceae) represent a classic example of the difficulty in understanding evolutionary relationships between tropical-temperate family pairs. In Apiales, this problem is further compounded by phylogenetic confusion at almost every taxonomic level, including ordinal, interfamilial, and infrafamilial, due largely to difficulties in understanding trends in morphological evolution. Phylogenetic analyses of rbcL sequences were employed to resolve relationships at the ordinal and familial levels. The results of the ordinal analysis confirm the placement of Apiales in an expanded subclass Asteridae as the sister group to Pittosporaceae, and refute the traditional alliance of Apiales with Cornales and Rosidae. This study has also resolved relationships of a number of enigmatic genera, suggesting, for example, that Melanophylla, Aralidium, Griselinia, and Toricellia are close relatives of Apiales. Clarification of phylogenetic relationships has concomitantly provided insights into trends of morphological evolution, and suggests that the ancestral apialean taxon was probably bicarpellate, simple-leaved, woody, and paleotropical. Phylogenetic analysis at the family level suggests that apiaceous subfamily Hydrocotyloideae, often envisioned as an intermediate group between Apiaceae and Araliaceae, is polyphyletic, with some hydrocotyloids closely allied with Araliaceae rather than Apiaceae. With the exception of some hydrocotyloids, Apiaceae appear to be monophyletic. The relationship between Apiaceae and Araliaceae remains problematic. Although the shortest rbcL trees suggest that Apiaceae are derived from within a paraphyletic Araliaceae, this result is only weakly supported.     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.