p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.     

3-Alkylthio-1,2,4-triazine dimers with potent antimalarial activity

We report on the discovery of 3-alkylthio-1,2,4-triazine dimers that are potently toxic to Plasmodium falciparum, with single digit nanomolar activity, and up to several thousand-fold lower toxicity to mammalian cells. They are equipotent against chloroquine-resistant strains of P. falciparum.     

Redox activity associated with the maturation and capacitation of mammalian spermatozoa

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.     

Circulating free nitrotyrosine in obstructive sleep apnea

Obstructive sleep apnea (OSA) has been increasingly linked to cardiovascular disease, endothelial dysfunction, and oxidative stress, generated by repetitive nocturnal hypoxemia and reperfusion. Circulating free nitrotyrosine has been reported as a novel biomarker of nitric oxide (NO)-induced oxidative/nitrosative stress. Nitrosative stress has been implicated as a possible mechanism for development of cardiovascular diseases. We tested the hypothesis that repetitive severe hypoxemia resulting from OSA would increase NO-mediated oxidative stress. We studied 10 men with newly diagnosed moderate to severe OSA who were free of other diseases, had never been treated for OSA, and were taking no medications. Nitrotyrosine measurements, performed by liquid chromatography-tandem mass spectrometry, were made before and after untreated apneic sleep. We compared free nitrotyrosine levels in these patients with those obtained at similar times in 10 healthy male control subjects without OSA, with similar age and body mass index. Evening baseline nitrotyrosine levels were similar before sleep in the control and OSA groups [0.16 +/- 0.01 and 0.15 +/- 0.01 ng/ml, respectively, P = not significant (NS)]. Neither normal nor disturbed apneic sleep led to significant changes of plasma nitrotyrosine (morning levels: control group 0.14 +/- 0.01 ng/ml; OSA group 0.15 +/- 0.01 ng/ml, P = NS). OSA was not accompanied by increased circulating free nitrotyrosine either at baseline or after sleep. This observation suggests that repetitive hypoxemia during OSA does not result in increased NO-mediated oxidative/nitrosative stress in otherwise healthy subjects with OSA.     

The porphobilinogen synthase catalyzed reaction mechanism

Porphobilinogen synthase (PBGS) catalyzes the first common reaction in the biosynthesis of the tetrapyrroles, the asymmetric condensation of two molecules of delta-aminolevulinic acid to form porphobilinogen. There is a variable requirement for an essential active site zinc that necessitates consideration of PBGS as an enzyme that may exhibit phylogenetic diversity in its chemical reaction mechanism. Recent crystal structures suggest reaction mechanisms that involve two covalent Schiff base linkages between adjacent active site lysine residues and each of the two substrate molecules. The reaction appears to stall at a covalently bound almost-product intermediate that is poised for breakdown to product upon binding of a substrate molecule to an adjacent active site and a subsequent conformational change.     

Allelic loss at the GPx-1 locus in cancer of the head and neck

Glutathione peroxidase is a selenium-containing, antioxidant enzyme previously implicated in the risk and development of lung and breast cancer, in part the result of allelic loss at the GPx-1 locus. This study examined allelic loss at the same locus in squamous cell carcinomas of the head and neck. The frequency of a polymorphism at codon 198 resulting in either a leucine or a proline at that position was surveyed by comparing 133 DNA samples obtained from head and neck tumors and 517 samples obtained from cancer-free individuals. Tumor DNAs exhibited fewer pro/leu heterozygotes as compared to DNA obtained from the cancer-free population. Fewer GPx-1 heterozygotes were verified by determining the frequency of highly polymorphic alanine repeat sequences in the same gene. The analysis revealed an approximately 42% reduction in heterozygosity in the DNA from the tumor samples. In order to assess loss of heterozygosity (LOH) at the GPx-1 locus, DNA was genotyped from peripheral lymphocytes, tumor tissue, and microscopically normal tissues adjacent to the tumor, derived from the same patients. These studies indicated LOH at the GPx-1 locus in each of the three tumor/normal tissues sample sets examined. Furthermore, LOH in the microscopically normal tissues at the tumor margin occurred in two of the three sample sets examined. These data implicate GPx-1 in the development of squamous cell carcinoma the head and neck and suggest that allelic loss of this gene, or one tightly linked to it, is an early event in the development of this type of malignancy. Author to whom all correspondence and reprint requests should be addressed. These authors contributed equally to this work.