Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences

The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.     

Effectiveness of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) against cerium toxicity

Therapeutic efficacy of EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) against cerium intoxicated mice was studied. Administration of cerium showed significant decrease in haemoglobin percentage, RBC counts and blood glucose level with an increase in the activity of serum transaminases and WBC counts. Decrease in the activity of alkaline phosphatase and glycogen content was noted in liver and kidney after cerium exposure. Light and electron microscopical investigations showed that these changes were recouped considerably with the administration of EGTA suggesting its therapeutic efficacy against cerium toxicity.     

Inhibitors of topoisomerases as anticancer drugs: problems and prospects

DNA topoisomerases, which solve topological problems associated with various DNA transactions, are the targets of many therapeutic agents. Various topoisomerase inhibitors especially, topo-poisons, camptothecin (topo-I) and etoposide (topo-II) are some of the drugs that are used in the current treatment protocols, particularly for the treatment of leukemia (AML, ALL etc). However, tumor resistance, normal and non-specific tissue cytotoxicity are the limitations for successful development of these drugs as one of the primary therapeutic agents for the treatment of tumors in vitro. This brief review presents the current understanding about cytotoxicity development and outlines various approaches to overcome the limitations for enhancing the efficacy of topo-poison based anticancer drugs.     

Changes in CD38 expression and ADP-ribosyl cyclase activity in rat myometrium during pregnancy: influence of sex steroid hormones

Cyclic ADP-ribose (cADPR), synthesized by CD38, regulates intracellular calcium in uterine smooth muscle. CD38 is a transmembrane protein that has both ADP-ribosyl cyclase and cADPR hydrolase enzyme activities involved in cADPR metabolism. CD38 expression and its enzyme activities in uterine smooth muscle are regulated by estrogen. In the present study, we examined CD38 expression, its enzyme activities, and cADPR levels in myometrium obtained from rats at 14-17 days of gestation (preterm) and at parturition (term). CD38 expression, ADP-ribosyl cyclase activity, and cADPR levels were higher in uterine tissues obtained from term rats compared with that of preterm rats, while activity of cADPR hydrolase did not significantly change. In an effort to address whether changes in estrogen: progesterone ratio that occur during pregnancy account for the observed effects on CD38 expression and function, we determined the effect of different doses of progesterone in the presence of estrogen on CD38 expression and its enzyme activities in uterine smooth muscle obtained from ovariectomized rats. In myometrium obtained from ovariectomized rats, estrogen administration caused increased CD38 protein expression and ADP-ribosyl cyclase activity. The estrogen-induced increases in CD38 expression and ADP-ribosyl cyclase activity were inhibited by simultaneous administration of 10 or 20 mg of progesterone. These results indicate that the estrogen:progesterone ratio determines CD38 expression and ADP-ribosyl cyclase activity. These changes in CD38/cADPR pathway may contribute to increased uterine motility and onset of labor.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

The comparative effects of metals on the hatching of earthworm cocoons

To establish the use of Metaphire posthuma as a sensitive model for ecotoxicological studies, the comparative effects of five metals on the hatching profiles of the cocoons of the earthworms, Metaphire posthuma, Eisenia foetida and Perionyx excavatus, were studied. The cocoons of the three species of earthworms were exposed to copper, chromium (III), chromium (VI), lead and zinc at 1.25, 2.5, 5.0 and 10.0 ppm. Viable cocoons were incubated at 20 +/- degrees C by using the immersion method. The results indicated that the inhibition of cocoon hatching was concentration dependent. The normal hatching, delayed hatching and non-viability of cocoons were recorded. At a concentration of 1.25 ppm, there was almost no effect on the hatching of the cocoons of all three species of earthworms, except when exposed to chromium (VI), but higher concentrations (2.5 and 5.0 ppm) caused severe effects. It was concluded that M. posthuma was more sensitive than the other two species, and that it is a suitable model for use in ecotoxicity testing.     

Neuroprotective effect of BDNF in young and aged 6-OHDA treated rat model of Parkinson disease

Brain derived neurotrophic factor (BDNF) has been shown to exert trophic effects on dopaminergic neurons against 6-hydroxydopamine (6-OHDA) in young rat. Since the degeneration of substantia nigra dopaminergic neurons that occurs in Parkinson's disease is more often than not confined to elderly individuals, it is of interest to determine whether the effects of BDNF against 6 hydroxydopamine (6-OHDA) in young rats can be extended to aged animals. 6-hydroxydopamine was stereotaxically injected into the striatum of young (3-months) and aged (24-months) rats, which were treated two hours earlier with BDNF. 6-OHDA results in almost complete destruction of substantia nigra pars compacta dopaminergic neurons. BDNF injection significantly changed apomorphine induced rotations from 132 +/- 15 to 181 +/- 10, staircase test from 73 +/- 2% to 61 +/- 3%, initiation time from 7 +/- 2 to 12 +/- 1 sec, and disengage time from 80 +/- 7 to 90 +/- 5 sec in young and aged animals, respectively. It is concluded that BDNF causes the limited behavior recovery of striatal DA systems from 6-OHDA toxicity in aged animals.     

Euler buckling-induced folding and rotation of red blood cells in an optical trap

We investigate the physics of an optically driven micromotor of biological origin. When a single, live red blood cell (RBC) is placed in an optical trap, the normal biconcave disc shape of the cell is observed to fold into a rod-like shape. If the trapping laser beam is circularly polarized, the folded RBC rotates. A model based on geometric considerations, using the concept of buckling instabilities, captures the folding phenomenon; the rotation of the cell is rationalized using the Poincaré sphere. Our model predicts that (i) at a critical power of the trapping laser beam the RBC shape undergoes large fluctuations, and (ii) the torque that is generated is proportional to the power of the laser beam. These predictions are verified experimentally. We suggest a possible mechanism for the emergence of birefringent properties in the RBC in the folded state.     

Stabilizing proteins from sequence statistics: the interplay of conservation and correlation in triosephosphate isomerase stability

Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution. Consensus mutations have proven effective for increasing stability, but these mutations are successful only about half the time. We set out to understand why some consensus mutations fail to stabilize, and what criteria might be useful to predict stabilization more accurately. Overall, consensus mutations at more conserved positions were more likely to be stabilizing in our model, triosephosphate isomerase (TIM) from Saccharomyces cerevisiae. However, positions coupled to other sites were more likely not to stabilize upon mutation. Destabilizing mutations could be removed both by removing sites with high statistical correlations to other positions and by removing nearly invariant positions at which "hidden correlations" can occur. Application of these rules resulted in identification of stabilizing mutations in 9 out of 10 positions, and amalgamation of all predicted stabilizing positions resulted in the most stable yeast TIM variant we produced (+8 °C). In contrast, a multimutant with 14 mutations each found to stabilize TIM independently was destabilized by 2 °C. Our results are a practical extension to the consensus concept of protein stabilization, and they further suggest the importance of positional independence in the mechanism of consensus stabilization.