Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

The p21Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct end-labelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, filling of the approximately 30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap filling was uncoupled from incision. Binding p21 to PCNA could also inhibit filling of model 30 nt gaps by both purified DNA polymerases delta and epsilon. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap filling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an efficient inhibitor of NER synthesis in cell extracts.     

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.     

Developmental and hormonal regulation of hepatocyte growth factor expression and action in the bovine ovarian follicle

BACKGROUND: Anorexia and cachexia are well-known sequelae of cancer that contribute to morbidity and mortality. In several studies in patients with non-hormone-sensitive tumors, synthetic progestogens were shown to exert beneficial effects on appetite and weight loss. The current study was undertaken to investigate the effects of medroxyprogesterone acetate (MPA) on food intake, body composition, and resting energy expenditure (REE). METHODS: Fifty-four patients with non-hormone-sensitive cancer, generally characterized by substantial weight loss and hypermetabolism, received either MPA, 500 mg, or placebo twice daily for 12 weeks (double-blind study). Food intake was measured by dietary history, body composition was assessed by deuterium dilution (fat mass, fat-free mass), and REE was obtained by indirect calorimetry. RESULTS: Compared with placebo, 12 weeks of MPA led to an increase in energy intake (between-group difference, 426 kcal/day; P = 0.01) that was significantly associated (r = 0.68, P = 0.003) with an increase in fat mass (between-group difference, 2.5 kg; P = 0.009). Fat-free mass was not significantly influenced. REE increased during MPA treatment: at 6 weeks, the between-group difference in change was 135 kcal/day (P = 0.009); after 12 weeks, this difference was 93 kcal/ day (P = 0.07). CONCLUSIONS: The authors conclude that MPA is able to stimulate increased food intake significantly and to reverse fat loss concomitantly in patients with non-hormone-sensitive cancer.     

Stereotypes and retrieval-provoked illusory source recollections.

When expectations and stereotypes are activated at retrieval, they spontaneously create distorted and illusory recollections that are consistent with these expectations. Participants studied doctor (physician)-related and lawyer-related statements that were presented by 2 different people. When informed, on a subsequent source memory test, (i.e., of who presented what) that one of the study sources was actually a doctor and the other source was a lawyer, there was a strong tendency to attribute the test items in a stereotype-consistent manner. In 3 experiments, participants frequently reported recollecting specific details, such as via "remember" judgments, to justify their stereotype-consistent but incorrect responses. These experiments rule out explanations involving either the misattribution of strong familiarity or differences in the bias to making remember responses as accounts for the illusory source attributions. Instead, the illusory recollections are consistent with the notion that recollective experience is manufactured from both the information in the memory trace and information in the retrieval environment, such as an individual's expectations, stereotypes, and general knowledge. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase

Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000. The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a [2Fe-2S]2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution, the reduced [4Fe-4S]+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the [4Fe-4S]cluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear [3Fe-4S] cluster or a [2Fe-2S] cluster. Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.     

Intravenous glucose after out-of-hospital cardiopulmonary arrest: a community-based randomized trial

Question: Does the common practice of infusing small amounts of glucose after cardiopulmonary arrest worsen neurologic outcome? Design and setting: A community-based randomized trial in Seattle, WA. Paramedics treated all patients with out-of-hospital cardiac arrest in a standard fashion except that the intravenous infusion did or did not contain glucose; ie, patients received either usual treatment, with 5% dextrose in water (D5W), or alternative, with half normal saline (0.45S). Outcomes: The main outcome was awakening, defined as the patient having comprehensible speech or following commands as determined by chart review. Other outcomes were survival to hospital admission and to discharge. Results: Over 2 years, paramedics randomized 748 patients. The type of fluid administered was not significantly related to awakening (16.7% for D5W versus 14.6% for 0.45S), admission (38.0% for D5W versus 39.8% for 0.45S), or discharge (15.1% for D5W versus 13.3% for 0.45S). As in previous studies, patients whose arrest had likely been on a cardiac basis with initial rhythms of ventricular fibrillation or asystole had admission blood glucose levels significantly related to awakening: mean = 309 mg/dl for never awakening and 251 mg/dl for awakening. Of note, the relation between glucose and awakening was reversed in the remaining patients, who had electromechanical dissociation or noncardiac mechanisms of arrest. Conclusion: Current practices of using limited amounts of glucose-containing solutions after cardiopulmonary arrest do not need to be changed. Blood glucose level on admission is a prognostic indicator but depends on the type of arrest.     

Automatic cognitions and teen dating violence.

Objective: The present research examined whether the level of aggression in automatic cognitions was positively associated with teen dating violence after accounting for more consciously controlled, self-reported attitudes about dating violence. Method: At baseline, 95 teens who had been remanded to the juvenile court system because of antisocial behavior completed a word-completion task designed to measure the level of aggression in their automatic cognitions. Teens also completed questionnaire measures of attitudes about dating violence and dating violence perpetration during the previous three months, and then provided data on dating violence perpetration every two weeks over a 3-month follow-up period. Results: The level of aggression in automatic cognitions was positively associated with dating violence perpetration after accounting for teens' self-reported attitudes about dating violence. This pattern of results emerged with both concurrent and prospective associations. It is noteworthy that aggression in automatic cognitions also predicted changes in dating violence perpetration over the 3-month follow-up period, even after controlling for baseline levels of the perpetration of dating violence and teens' self-reported attitudes about dating violence. Conclusions: These findings suggest that theoretical models of teen dating violence should consider the role of automatic as well as more consciously controlled cognitive processes in the perpetration of teen dating violence. In addition, clinical efforts to reduce teen dating violence might benefit from targeting automatic as well as more controlled cognitive processes. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Aging, metamemory, and high-confidence errors: A misrecollection account.

Two experiments showed that older adults were worse than younger adults at judging the accuracy of their responses on source identification (i.e., who said what) and cued-recall tests. It is important to note that this age-related metamonitoring impairment occurred even after older and younger adults were matched on overall source accuracy and cued-recall accuracy. By contrast, older and younger adults showed comparable metamonitoring capacities when assessing the likely accuracy of old-new recognition judgments and responses to questions about general knowledge. These experiments are consistent with the misrecollection account of cognitive aging, which suggests that age-related memory impairments are due to older adults' vulnerability to making high-confidence errors when answering questions that require memory for specific details about recently learned events. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Traumatic rupture of the thoracic aorta during the second trimester of pregnancy

A 36-year-old woman in the second trimester of pregnancy underwent emergent operative repair of a traumatic aortic disruption caused by a motor vehicle accident. Left atrial-to-femoral artery bypass was used to maintain fetal circulation during the cross-clamp period. Her healthy, full-term child was subsequently delivered 3 months later by normal vaginal delivery.