Phosphatases join kinases in DNA-damage response pathways

An inappropriate imbalance of kinase and phosphatase activities could be deleterious to cellular processes such as proliferation. Cellular responses to DNA damage use signal-transduction pathways involving phosphorylation events, and such modifications must be reversible to make these responses transient, rather than permanent, events. Three recent articles describe roles for two phosphatases in signaling pathways that are activated after DNA damage.     

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

Background  

In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.     

Comparison of PCR and clinical laboratory tests for diagnosing <Emphasis Type="Italic">H. pylori</Emphasis> infection in pediatric patients

Background

Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results

Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions

The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

     

Fragments of ATM which have dominant-negative or complementing activity.

The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogated the S-phase checkpoint after ionizing irradiation and enhanced radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complemented radiosensitivity and the S-phase checkpoint and reduced chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM.     

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.     

Solvent-induced free energy landscape and solute-solvent dynamic coupling in a multielement solute

Molecular dynamics simulations using a simple multielement model solute with internal degrees of freedom and accounting for solvent-induced interactions to all orders in explicit water are reported. The potential energy landscape of the solute is flat in vacuo. However, the sole untruncated solvent-induced interactions between apolar (hydrophobic) and charged elements generate a rich landscape of potential of mean force exhibiting typical features of protein landscapes. Despite the simplicity of our solute, the depth of minima in this landscape is not far in size from free energies that stabilize protein conformations. Dynamical coupling between configurational switching of the system and hydration reconfiguration is also elicited. Switching is seen to occur on a time scale two orders of magnitude longer than that of the reconfiguration time of the solute taken alone, or that of the unperturbed solvent. Qualitatively, these results are unaffected by a different choice of the water-water interaction potential. They show that already at an elementary level, solvent-induced interactions alone, when fully accounted for, can be responsible for configurational and dynamical features essential to protein folding and function.     

DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture

Significant progress has been made in recent years in elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. Much of our understanding of the mechanisms involved in cellular DNA damage response pathways has come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. Ataxia-telangiectasia mutated (ATM), the gene mutated in the disorder ataxia-telangiectasia, codes for a protein kinase that is a central mediator of responses to DNA double-strand breaks (DSB) in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the response of the cell to the DNA damage. We recently developed a novel system to create DNA DSBs at defined endogenous sites in the human genome and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation. Results from this system showed the functional importance of ATM kinase activity and phosphorylation in the response to DSBs and supported a model in which ordered chromatin structure changes that occur after DNA breakage and that depend on functional NBS1 and ATM facilitate DNA DSB repair. Insights about these pathways provide us with opportunities to develop new approaches to benefit patients. Examples and opportunities for developing inhibitors that act as sensitizers to chemotherapy or radiation therapy or activators that could improve responses to cellular stresses, such as oxidative damage, are discussed. Relevant to the latter, we have shown benefits of an ATM activator in disease settings ranging from metabolic syndrome to cancer prevention.