DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes.

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.

Conserved structural motifs governing the stoichiometric repair of alkylated DNA by O(6)-alkylguanine-DNA alkyltransferase.

O(6)-alkylguanine-DNA alkyltransferase (AGT) directly repairs alkylation damage at the O(6)-position of guanine in a unique, stoichiometric reaction. Crystal structures of AGT homologs from the three kingdoms of life reveal that despite their extremely low primary sequence homology, the topology and overall structure of AGT has been remarkably conserved. The C-terminal domain of the two-domain, alpha/beta fold bears a helix-turn-helix (HTH) motif that has been implicated in DNA-binding by structural and mutagenic studies. In the second helix of the HTH, the recognition helix, lies a conserved RAV[A/G] motif, whose "arginine finger" promotes flipping of the target nucleotide from the base stack. Recognition of the extrahelical guanine is likely predominantly through interactions with the protein backbone, while hydrophobic sidechains line the alkyl-binding pocket, as defined by product complexes of human AGT. The irreversible dealkylation reaction is accomplished by an active-site cysteine that participates in a hydrogen bond network with invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. Structural and biochemical results suggest that cysteine alkylation opens the domain-interfacing "Asn-hinge", which couples the active-site to the recognition helix, providing both a mechanism for release of repaired DNA and a signal for the observed degradation of alkylated AGT.

Functional impairment, depression, and life satisfaction among older hemodialysis patients and age-matched controls: a prospective study.

OBJECTIVE: To compare change over time in functional impairment, depression, and life satisfaction among older dialysis patients and age-matched controls. DESIGN: Prospective cohort study over 3 years. SETTING: Urban and rural communities throughout Georgia. SUBJECTS: One hundred thirteen prevalent renal failure patients on in-center hemodialysis and 286 controls. MAIN OUTCOME MEASURES: Ordinal functional impairment index and life satisfaction rating, and Center for Epidemiologic Studies Depression Scale. RESULTS: Dialysis patients, compared with controls, reported significantly more functional impairment at baseline, and also at follow-up after adjusting for baseline impairment and covariates. Dialysis patients had higher depression scores at baseline, and also at follow-up after adjusting for baseline depression and covariates. In contrast, dialysis patients reported lower life satisfaction at baseline than did controls, but the two cohorts were not significantly different on reported life satisfaction at follow-up, after adjusting for baseline life satisfaction and race. In both cohorts, functional impairment and depression were significantly related. CONCLUSION: Older dialysis patients' life satisfaction at a 3-year follow-up, which was similar to life satisfaction among age-matched controls, indicates the value of delivered dialysis care; the value of this care would be increased by reducing excess functional impairment in these patients.

Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding.

Human O(6)-alkylguanine-DNA alkyltransferase (AGT), which directly reverses endogenous alkylation at the O(6)-position of guanine, confers resistance to alkylation chemotherapies and is therefore an active anticancer drug target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes reveal an unexpected zinc-stabilized helical bridge joining a two-domain alpha/beta structure. An asparagine hinge couples the active site motif to a helix-turn-helix (HTH) motif implicated in DNA binding. The reactive cysteine environment, its position within a groove adjacent to the alkyl-binding cavity and mutational analyses characterize DNA-damage recognition and inhibitor specificity, support a structure-based dealkylation mechanism and suggest a molecular basis for destabilization of the alkylated protein. These results support damaged nucleotide flipping facilitated by an arginine finger within the HTH motif to stabilize the extrahelical O(6)-alkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cysteine alkylation sterically shifts the HTH recognition helix to evidently mechanistically couple release of repaired DNA to an opening of the protein fold to promote the biological turnover of the alkylated protein.

Occupational risk factors for sarcoma subtypes.

Herbicides, chlorophenols, and other occupational exposures are suspected risk factors for soft-tissue sarcoma, but the epidemiologic evidence is inconsistent. Given that soft-tissue sarcomas represent a heterogeneous mix of cancer subtypes and that these subtypes have different disease patterns by race, sex, and age at diagnosis, studying all soft-tissue sarcomas combined may mask subtype-specific associations. Using the Selected Cancers Study, a large population-based case-control study of sarcoma conducted among U.S. men aged 30 to 60 in 1984 to 1988, we explored the occupational risk factors for soft-tissue sarcoma subtypes and skeletal sarcoma. The analysis included 251 living sarcoma cases (48 dermatofibrosarcoma protuberans, 32 malignant fibrohistiocytic sarcoma, 67 leiomyosarcoma, 53 liposarcoma, and 51 skeletal sarcoma) and 1908 living controls. Exact conditional logistic regression models suggested patterns of subtype specificity for occupational exposures. Self-reported herbicide use was associated with malignant fibrohistiocytic sarcoma (OR = 2.9, 95% CI = 1.1-7.3). We found elevated risks for chlorophenol exposure and cutting oil exposure and malignant fibrohistiocytic sarcoma and leiomyosarcoma. We found no occupational risk factor for liposarcoma. Polytomous regression models identified different odds ratios across subtypes for plywood exposure and exposure to wood and saw dust. Although exploratory, this analysis suggests that occupational risk factors for sarcoma are not uniform across subtypes.

Adhesion and motility of polymorphonuclear leukocytes isolated from the blood of rats exposed to ozone: potential biomarkers of toxicity.

Ozone (O3) exposure of rats results in airway epithelial injury and an infiltration of polymorphonuclear leukocytes (PMNs) into the lungs, suggesting alteration of PMN functions. To identify the altered PMN functions and their possible effects on epithelia, rats were exposed to air or 0.8 ppm O3 for 2 hr. PMNs were isolated from the blood and incubated with an epithelial cell line derived from rat lung (ARL-14) or primary alveolar type II cell cultures. The PMNs from the O3-exposed rats exhibited stimulated motility and spontaneous redistribution of actin filaments and adhered in a greater number to the epithelial cells when compared with the PMNs from the air-exposed rats. Actin caps usually formed at the sites of contact between the PMNs and epithelial cells, suggesting a cytoskeletal role in the inflammatory-epithelial cell interaction. By scanning electron microscopy, PMNs from air-exposed rats had features of non-motile cells. In a striking contrast to this, PMNs from O3-exposed rats revealed surface modifications, which were quite prominent at the sites of PMN-epithelial cell contacts. Despite these morphological changes, the PMNs from O3-exposed rats did not alter the epithelial resistance, a measure of paracellular permeability. In contrast to this, PMNs stimulated by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine not only exhibited greater adhesion to the epithelial cells, but also caused a reduction in epithelial resistance. The changes reflecting altered morphology, motility, and adhesion of PMNs from O3-exposed rats may represent important steps in the O3-induced inflammatory response that precedes barrier disruption in vivo, but they are not associated with increased epithelial permeability in an in vitro system. Besides their mechanistic relevance, the alterations of vascular PMNs may serve as important biomarkers for detecting O3 effects.

Evaluation of cisplatin and DTIC in inoperable stage III and IV melanoma. A Southwest Oncology Group study.

The Southwest Oncology Group entered 62 patients with Stage IV or inoperable Stage III (one patient) melanoma into SWOG protocol 8804 and treated them with cisplatin 100 mg/m2 and DTIC 750 mg/m2 i.v. infusion over 15-30 minutes. There were 18 patients with brain metastases and four ocular primaries. Five patients, all without bain metastases, were ineligible. Responses of 8 patients could not be determined, and 11 patients received only one course of treatment. Of the eligible patients, 46 (81%) had some hematologic toxicities, with 31 of these (67%) having grade III or worse. There were 23 patients (40%) with renal toxicities. The miscellaneous toxicities were muscle weakness, flu-like symptoms, and fatigue. Five patients died while on treatment. There were no complete responses. Eight patients had partial responses ranging from 1.5 to 10.5 months, although two patients were still alive at 30.4 and 30.9 months. The estimated response rate for patients with brain metastases was 11%. The estimated response rate for patients without brain metastases was 13%. If one unconfirmed partial response is included, the overall response rate is 14% with a 95% confidence interval of 6% to 26%. It is concluded that DTIC and cisplatin have definite activity in melanoma, but, at least in this population, the toxicity is treatment-limiting and requires close attention to patient care.

Attenuation of ozone-induced airway permeability in rats by pretreatment with cyclophosphamide, FPL 55712, and indomethacin.

Exposure of rats to ozone (O3) produces an increase in airway permeability and a concomitant influx of polymorphonuclear leukocytes in the lung. These observations raise the possibility that the inflammatory cells play a role in the cellular injury and increased airway permeability after O3 exposure. This study was therefore designed to determine if the inflammatory cells or their products are essential for the O3 effect. In a series of experiments, rats were rendered leukopenic with cyclophosphamide, treated with leukotriene B4 (LTB4), or with the inhibitors of lipoxygenase or cyclooxygenase products of arachidonic acid, followed by exposure to O3. A 2-h exposure to 0.8 ppm O3 caused a significant increase in the flux of proteins and albumin in bronchoalveolar lavage (BAL) and elevated the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood. The treatment with cyclophosphamide caused a significant reduction in the circulating and pulmonary leukocytes and prevented an increase in tracheal mucosal permeability to 99mTc-DTPA and the protein and albumin flux in BAL. While the intratracheal instillation of LTB4 did not affect the permeability, tracheal permeability and albumin levels in BAL in rats treated with LTD4 antagonist FPL 55712 and exposed to O3 were lower than in the untreated O3-exposed rats. Pretreatment with indomethacin also prevented the O3 effects, as reflected by the decreased protein and albumin flux in BAL and 99mTc-DTPA transport from trachea to blood. These data show a reduction in the effect of O3 by agents that affect leukocytes or their products. The results support a mechanism of increased permeability that is dependent upon inflammatory cells and their products.

Amalgam tattoo: report of an unusual clinical presentation and the use of energy dispersive X-ray analysis as an aid to diagnosis.

An unusual appearing gingival amalgam pigmentation (amalgam tattoo) that completely surrounded the maxillary right first premolar in a 13-year-old boy is presented. Because of the wide distribution and apparent clinical progression of the discoloration, an excisional biopsy was performed. The histopathologic diagnosis of amalgam pigmentation was confirmed in paraffin sections by energy dispersive X-ray microanalysis. Silver, tin, and mercury were detected in the specimen.

Ameliorating the impact of teen-age pregnancy on parent and child.

The increase in teen-age pregnancy creates a growing population of young mothers unable to care adequately for themselves or their children. Responding to budget cuts in Utah, the authors report on a program that combines the resources of an agency and a university program to teach mothers child care and self-sufficiency, promotes the healthy development of their children, and gives field training to social work students.

PIP: This article reports on a program that combines the resources of an agency and a university program to teach mothers child care and self sufficiency, promotes the healthy development of their children, and gives field training to social work students. The program was a response to budget cuts in Utah and awareness that the increase in teengage pregnancy creates a growing population of young mothers unable to care adequately for themselves or their children. The increase is attributed to a drop in the age of menarche and a steady increase in the number of sexually active young people. In Utah, where conservative mores concerning sexuality predominate, teenage unmarried mothers frequently perceive themselves as alienated from the mainstream of society and human service agencies have been unable to deal adequately with the increased demands for service. The Single Parent Project combines the resources of the Children's Aid Society and the Early Childhood Research Program at Utah State University to help pregnant adolescents and their at risk preschool children. In 1980-81, a core group of 15 mothers and 29 children were serviced. Mothers had to meet 4 criteria from a list that included health problems, economic privation, substance abuse, age factors, deficient parenting, negative or no support network and previous victim of abuse. Children had to meet two or more criteria including birth problems, nutrition, developmental and behavioral problems or reported victim of abuse or neglect. The program included weekly group sessions with mothers, and home intervention using individualized plans for each child. Activities and resources used are described. Success is measured in a comparison with non program users. A significantly higher percentage of program graduates found employment, went on to college, had less dependence on welfare and fewer referrals for child abuse. The programs next phase calls for an experimental design to test the program's effectiveness.