Biochemical characterization of the small isoform of Drosophila melanogaster RECQ5 helicase.

Recently the gene encoding a member of the RecQ helicase family, RecQ5, was cloned from the fruit fly, Drosophila melanogaster [J.J.Sekelsky, M.H.Brodsky, G.M. Rubin and R.S. Hawley (1999) Nucleic Acids Res., 27, 3762-3769]. The Drosophila RecQ5 transcript is alternatively spliced, like its human counterpart, to yield three protein isoforms. Two of these isoforms are almost identical and have a predicted molecular weight of 54 kDa. The third isoform is larger and contains, in addition to the helicase domain shared by all three isoforms, a long highly charged C-terminal region. A small isoform of the Drosophila RecQ5 protein (RECQ5) has been expressed in Escherichia coli and purified. The purified protein is a single-stranded DNA-stimulated ATPase (dATPase) and a 3'-->5' DNA helicase. Hydrolysis of the nucleotide cofactor is required for unwinding activity and dATP supported the unwinding reaction better than other NTPs. The turnover number for the single-stranded DNA-stimulated dATPase activity was 1380 min(-1), approximately 1.5-fold higher than that observed for the ATPase activity (900 min(-1)). The purified protein catalyzed unwinding of partial duplex substrates up to at least 93 bp, however, unwinding of an 89 bp blunt duplex substrate was not detected.

DNA helicases of Escherichia coli.

A great deal has been learned in the last 15 years with regard to how helicase enzymes participate in DNA metabolism and how they interact with their DNA substrates. However, many questions remain unanswered. Of critical importance is an understanding of how NTP hydrolysis and hydrogen-bond disruption are coupled. Several models exist and are being tested; none has been proven. In addition, an understanding of how a helicase disrupts the hydrogen bonds holding duplex DNA together is lacking. Recently, helicase enzymes that unwind duplex RNA and DNA.RNA hybrids have been described. In some cases, these are old enzymes with new activities. In other cases, these are new enzymes only recently discovered. The significance of these reactions in the cell remains to be clarified. However, with the availability of significant amounts of these enzymes in a highly purified state, and mutant alleles in most of the genes encoding them, the answers to these questions should be forthcoming. The variety of helicases found in E. coli, and the myriad processes these enzymes are involved in, were perhaps unexpected. It seems likely that an equally large number of helicases will be discovered in eukaryotic cells. In fact, several helicases have been identified and purified from eukaryotic sources ranging from viruses to mouse cells (4-13, 227-234). Many of these helicases have been suggested to have roles in DNA replication, although this remains to be shown conclusively. Helicases with roles in DNA repair, recombination, and other aspects of DNA metabolism are likely to be forthcoming as we learn more about these processes in eukaryotic cells.

Duplex DNA binding activity of the avian myeloblastosis virus DNA polymerase.

Purified avian myeloblastosis virus DNA polymerase has a strong binding affinity for closed circular double-stranded DNA with no 3'-hydroxy termini. Because of this affinity the DNA polymerase can retain labeled native ColE1 DNA on nitrocellulose filters. When the reaction contains four enzyme molecules per ColE1 molecule about 50% of the DNA is retained. Higher enzyme: DNA ratios cause retention of nearly 100% of the DNA. The binding activity comigrates with DNA synthetic activity through ion-exchange chromatography and glycerol gradient centrifugation, in indication that it is an intrinsic activity of the DNA polymerase. Escherichia coli DNA polymerase I and bacteriophage T4 DNA polymerase do not show this binding activity, which suggests that it is not a common property of DNA polymerases. A novel application of enzyme kinetics using endonuclease-treated DNA reveals the relative quantities of enzyme molecules which are synthesizing at 3'-termini vs. molecules bound to double-stranded regions. Heat stability measurements indicate that the polymerizing activity of the enzyme can be almost completely eliminated while about half of the original binding activity is retained.

Site-directed mutations in motif VI of Escherichia coli DNA helicase II result in multiple biochemical defects: evidence for the involvement of motif VI in the coupling of ATPase and DNA binding activities via conformational changes.

Two site-directed mutants of Escherichia coli DNA helicase II (UvrD) were constructed to examine the functional significance of motif VI in a superfamily I helicase. Threonine 604 and arginine 605, representing two of the most highly conserved residues in motif VI, were replaced with alanine, generating the mutant alleles uvrD-T604A and uvrD-R605A. Genetic complementation studies indicated that UvrD-T604A, but not UvrD-R605A, functioned in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. Both mutant enzymes were purified and single-stranded DNA (ssDNA)-stimulated ATP hydrolysis, duplex DNA unwinding, and ssDNA binding were studied in the steady-state and compared to wild-type UvrD. UvrD-T604A exhibited a serious defect in ssDNA binding in the absence of nucleotide. However, in the presence of a non-hydrolyzable ATP analog, DNA binding was only slightly compromised. Limited proteolysis experiments suggested that UvrD-T604A had a "looser" conformation and could not undergo conformational changes normally associated with ATP binding/hydrolysis and DNA binding. UvrD-R605A, on the other hand, exhibited nearly normal DNA binding but had a severe defect in ATP hydrolysis (kcat=0.063 s-1 compared to 162 s-1 for UvrD). UvrD-T604A exhibited a much less severe decrease in ATPase activity (kcat=8.8 s-1). The Km for ATP for both mutants was not significantly changed. The results suggest that residues within motif VI of helicase II are essential for multiple biochemical properties associated with the enzyme and that motif VI is potentially involved in conformational changes related to the coupling of ATPase and DNA binding activities.

A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II. A biochemical and genetic characterization.

A site-specific lysine to methionine mutation has been engineered at the invariant Lys35 residue in the ATPase A binding site of the Escherichia coli uvrD gene encoding DNA helicase II. The mutant protein (UvrDK35M) has been purified to apparent homogeneity and characterized. The kcat for DNA-dependent ATP hydrolysis was less than 0.5% that of the wild-type enzyme with no change in the apparent Km for ATP. No unwinding of partial duplex DNA substrates could be detected using the mutant protein. Moreover, the mutant protein inhibited the unwinding reaction catalyzed by the wild-type protein at ratios of mutant enzyme to wild-type enzyme < 1. We conclude that the K35M mutation renders helicase II catalytically inactive as a DNA helicase with little or no effect on the ability of the enzyme to bind ATP, DNA, or other proteins. In vivo complementation assays indicate that the mutant protein cannot substitute for the wild-type protein in methyl-directed mismatch repair, suggesting that the ATPase and/or helicase activity of helicase II is required in this repair pathway. Additional genetic characterization of the uvrDK35M allele, supplied on a plasmid, suggests that expression of the mutant protein, at levels equivalent to that of the wild-type protein, results in a dominant negative phenotype. Expression of lower levels of the mutant protein, both in the presence and absence of wild-type helicase II, results in a constitutive induction of the cellular SOS response and extensive filamentation of cells. This induction of the SOS response is not due to a defect in methyl-directed mismatch repair. Taken together, these data are consistent with the notion that E. coli helicase II may have a role in DNA replication.

Genetic analysis of delta helD and delta uvrD mutations in combination with other genes in the RecF recombination pathway in Escherichia coli: suppression of a ruvB mutation by a uvrD deletion.

Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background, we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD ant the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and regN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Surprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the deltauvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two "RecF" homologous recombination pathways operating in a recBCsbcB(C) strain background.

Improving pediatric residents' alcohol and other drug use clinical skills: use of an experiential curriculum.

OBJECTIVE: To evaluate the effectiveness of an experiential alcohol and other drug curriculum on pediatric residents' knowledge, attitudes, and skills in alcohol and other drug (AOD) issues. DESIGN: Nonrandomized control trial. SETTING: Two university pediatric residency programs. PARTICIPANTS: Pediatric residents (n = 44). INTERVENTION: Intervention residents received an experiential AOD curriculum consisting of participation in an adolescent assessment program, interactive didactic sessions, role-playing practice, and interviewing skills sessions. The control group received no formal training. MAIN OUTCOME MEASURES: Pretesting and posttesting each group using written and Objective Structured Clinical Examination evaluations using standardized patients. Evaluations were videotaped and scored by an expert panel using a standardized scoring process. RESULTS: Pretest comparisons of written knowledge and clinical skills as assessed by the Objective Structured Clinical Evaluation showed no significant differences between the intervention and the control groups. Analysis of written test scores revealed that residents' general knowledge as well as knowledge of screening techniques and management resources related to AOD issues increased significantly more for the intervention group than for the control group from pretest to posttest (P < .001). Evaluation of the videotapes showed significant improvement for the intervention group compared with controls in overall score and in the use of specific screening techniques and interviewing skills (P < .05). Self-assessment of residents' interest, confidence, and competence in AOD issues improved significantly for intervention residents vs controls (P < .05). CONCLUSIONS: Pediatric residents receiving an experiential AOD curriculum increased their knowledge and clinical skills in AOD issues significantly more than residents receiving no formal training. Similar curricula and evaluation could be used by other primary care residency programs and could be implemented in other areas of adolescent health risk behaviors.

Intra-urban differences in breast cancer mortality: a study from the city of Malmö in Sweden.

STUDY OBJECTIVE: To assess whether in an urban population stage at breast cancer diagnosis is related to area of living and to what extent intra-urban differences in breast cancer mortality are related to incidence respectively stage at diagnosis. DESIGN: National registries were used to identify cases. Mortality in 17 residential areas was studied in relation to incidence and stage distribution using linear regression analysis. Areas with high and low breast cancer mortality, incidence and proportion of stage II+ tumours at diagnosis were also compared in terms of their sociodemographic profile. SETTING: City of Malmö in southern Sweden. PATIENTS: The 1675 incident breast cancer cases and 448 deaths that occurred in women above 45 years of age in Malmö 1986-96. MAIN RESULTS: Average annual age standardised breast cancer mortality ranged between residential areas, from 35/10(5) to 107/10(5), p = 0.04. Mortality of breast cancer was not correlated to incidence, r = 0.22, p = 0.39. The ratio of stage II+/0-I cancer incidence varied between areas from 0.45 to 1.99 and was significantly correlated to breast cancer mortality, r = 0.53, p = 0.03. Areas with high proportion of stage II+ cancers and high mortality/incidence ratio were characterised by a high proportion of residentials receiving income support, being foreigners and current smokers. CONCLUSIONS: Within this urban population there were marked differences in breast cancer mortality between residential areas. Stage at diagnosis, but not incidence, contributed to the pattern of mortality. Areas with high proportion of stage II+ tumours differed unfavourably in several sociodemographic aspects from the city average.

Building an institutional commitment to pain management in long-term care facilities.

This report reviews the development, implementation and findings of an inter-institutional study directed to the goal of making pain management an institutional priority in Wisconsin long-term care facilities. A total of 87 facilities were recruited in two training cohorts. Each facility identified a team of individuals, with responsibility and authority for care policies within their institution, to participate in four structured educational programs. Each team completed an Action Plan, structured around 14 national practice indicators of an institutional commitment to pain management. At baseline, 14% of facilities had > 51% of the indicators in place; at conclusion 74% of facilities had > 51% of indicators in place. This education project was successful across training cohorts at implementing critical pain management target indicators.

Assessment and treatment of discomfort for people with late-stage dementia.

People with dementia have often been excluded from pain studies. However, there is evidence supporting that people with dementia experience frequent pain, often poorly assessed and undertreated, and that the etiology for pain descriptions is poorly documented. The Assessment of Discomfort in Dementia (ADD) Protocol is designed to: a) more accurately assess discomfort in people with dementia who can no longer verbally describe physical pain or affective discomfort; b) more accurately and thoroughly treat physical pain and affective discomfort; and c) decrease inappropriate use of psychotropic medication. The use of the ADD Protocol was studies with a convenience sample of 104 residents of long-term care with end-stage dementia. Use of the ADD Protocol was associated with a significant decrease in discomfort (t = 6.56, p = 0.000). The most frequently seen behavioral symptoms associated with discomfort were tense body language, sad facial expression, fidgeting, perseverant verbalizations, and verbal outburts. The ADD Protocol was also associated with a significant increase in the use of scheduled analgesics and non-pharmacological comfort interventions. The protocol was not associated with an increase in the use of prn analgesics or with prn or scheduled psychotropics. This study has provided some support for the notion that the needs of people with significant dementia can be discerned and treated.

Approach to assessing adolescents on serious or sensitive issues.

For some young people, adolescence is a time of experimentation with high-risk behaviors that can lead to significant morbidity and mortality. Providers of adolescent health care need to become comfortable screening their patients for involvement with sexual activity, drug and alcohol use, violence, and mental health issues. This includes taking into consideration not only the patient's age and development status, but their rights to confidentiality and comprehensive health care. Several tools such as HEADSS interview and the CAGE questionnaire can easily be used to screen for high-risk behaviors. By taking the time to get to know adolescent patients, it is possible to identify problems early and intervene while the adolescent is amenable to treatment.

Physical findings and symptoms of depot medroxyprogesterone acetate use in adolescent females.

STUDY OBJECTIVE: To document physical and symptomatic changes in adolescent females using depot medroxyprogesterone acetate (DMPA) for contraception. DESIGN: A 30-month prospective experimental study using a convenience sample of subjects. SETTING: A pediatric primary care clinic in Milwaukee, Wisconsin that serves an urban population of low socioeconomic status. PARTICIPANTS: A mostly African-American group of 53 patients (mean age 16.5 +/- 1.3 years). INTERVENTIONS: Subjects received 150 mg DMPA intramuscularly in either the deltoid or gluteus muscle. The first two DMPA injections were given 6-8 weeks apart in an effort to decrease menstrual irregularity. Subsequent injections were given every 3 months. A questionnaire was administered at each visit to document physical and symptomatic changes. MAIN OUTCOME MEASURES: Included weight change, frequency and amount of menstrual bleeding, and perceived side effects and satisfaction both documented with a 5-point Likert scale. RESULTS: At 5, 11, and 17 months of DMPA use, 75%, 40%, and 19% of subjects continued DMPA. The most commonly perceived side effects were weight gain (27%), headache (25%), irregular periods (24%), fatigue (23%), abdominal pain (18%), and decreased sexual desire (15%). Significant weight gain was noted with an average increase of 6.0 +/- 6.0 kg at 11 months of DMPA use and 9.0 +/- 5.4 kg at 17 months. No menstrual bleeding was experienced by 30%-40% of adolescents in any 3-month injection period, and those who bled averaged 8-13 days of bleeding between injections. No pregnancies occurred and 87% of patients were either satisfied or very satisfied with DMPA as a method of contraception. CONCLUSION: Despite the documented side effects, DMPA is an effective, acceptable contraceptive for some adolescent females who are at high risk for pregnancy.

Psychological functioning and family discord among African-American adolescent females with and without a history of childhood sexual abuse.

OBJECTIVES: The aim was to examine the rate of childhood sexual abuse along with the factors (age of abuse onset, type of perpetrator, and duration of the abuse), as well as the relationship of these factors to psychological functioning among females with a history of childhood abuse. Second, to determine whether levels of psychological functioning and family discord differ among females with and without a history of childhood sexual abuse. METHOD: A cross-sectional design was used. Two hundred and forty-nine adolescent females were recruited from a community-based health program. Two trained female interviewers administered an anonymous survey that assessed childhood sexual abuse, psychological functioning, and family environment. RESULTS: Fifty-seven (22.9%) of those surveyed reported childhood sexual abuse, of which 44.3% were intrafamilial and 55.7% were interfamilial. Age at onset ranged from 3 years to 17 years; 62.5% reported that the sexual abuse occurred 1 to 4 times; 27.9% reported a duration ranging from 1 year to 13 years; and 9.6% reported a duration of 1 month to 7 months. Multiple regression analysis revealed that a greater duration predicted higher levels of depression and lower levels of self-esteem among females with a history of sexual abuse. Females with a history of childhood sexual abuse scored significantly lower on measures of self-esteem and mastery, and significantly higher on measures of physical and emotional abuse. CONCLUSIONS: Results indicate that adolescent females with a history of childhood sexual abuse suffer an array of negative sequelae that include psychological and family distress.

Relationship between scholastic and health behaviors and reading level in adolescent females.

This was a study of 102 adolescent females, 12-20 years of age, presenting to a central city clinic for medical care. Participants completed an oral questionnaire that included demographics, and questions regarding scholastic history, sexual behavior, and substance use. Each subject completed the Accuracy Level Test (ALT), a reading test. The subject's reading test grade level was subtracted from her appropriate grade in school to give a reading delay level (RDL). The mean reading grade level for all subjects was 6.7 +/- 2.6 and the average reading delay was 4.5 +/- 2.5 grades. Poor school attenders had greater reading delays (5.8 +/- 3.4 grades behind vs. 4.3 +/- 2.2 for good attenders p < 0.04), and those who repeated grades were also significantly delayed (5.5 +/- 2.4 grades behind vs. 3.7 +/- 2.3, p < 0.0007). Previously pregnant students had a greater delay in reading level than their nonpregnant peers (5.2 +/- 2.3 vs. 3.9 +/- 2.5 respectively p < 0.01). Delayed reading levels appear to correlate with some risk behaviors. The ALT may serve as a tool to identify high-risk patients who need more intensive clinical intervention.

Early detection and treatment of sexually transmitted disease in pregnant adolescents of low socioeconomic status.

This study evaluated the prevalence of sexually transmitted disease (STD) in adolescents presenting to a primary pediatric care clinic (PPCC) for the diagnosis of pregnancy and our ability to eradicate identified infections. We followed 168 pregnant adolescents of low socioeconomic status from their original pregnancy diagnosis until their first prenatal clinic visit. We collected screening cervical cultures for Neisseria gonorrhoeae and Chlamydia trachomatis by completing a pelvic examination on 91 patients at our PPCC. At the PPCC visit, 29% were positive for gonorrhea, chlamydia, or both. Screening tests for these infections were collected on all patients at the initial prenatal clinic visit. The risk for presenting to the prenatal clinic with a STD was significantly greater in patients not screened and treated for STD at the PPCC. Average delay from diagnosis to first prenatal clinic visit was 35.7 days. Thus, in this adolescent population, primary care providers are missing an important therapeutic opportunity by failing to identify and treat STD at initial diagnosis of pregnancy.

PIP: Maternal sexually transmitted disease (STD) is an important and preventable cause of infant morbidity and mortality. The early identification and treatment of STDs could, however, reduce the number of premature deliveries, low-birthweight infants, and neonatal deaths. Sexually active adolescents of low socioeconomic status (SES) are at increased risk for perinatal morbidity and mortality due to their substantially higher STD rates compared to adult women. Despite these facts, many primary care providers simply diagnose pregnancies with urine tests, then refer adolescents to a prenatal program for a thorough evaluation, including a screen for STDs. This practice means that young women infected with STDs at the diagnosis of pregnancy will most likely remain infected until they return to begin prenatal care and are subsequently diagnosed and treated for the problem. This study followed a group of pregnant adolescents from the initial diagnosis of pregnancy at a primary pediatric care clinic (PPCC) until the initial prenatal clinic visit. The study was undertaken to document the frequency of STD in adolescents presenting at such a PPCC for pregnancy diagnosis and to evaluate the ability to treat the infections once they are identified. 235 pregnancies were identified at the PPCC serving urban adolescents of low SES in Milwaukee, Wisconsin, over the period August 1, 1988, to January 31, 1992. 44 of these patients were seen at other prenatal programs, nine had miscarriages and nine had induced abortions before starting prenatal care, and five met the exclusion criteria, so the study findings pertain to only 168 subjects. 91 subjects were screened at the initial visit to the PPCC for gonorrhea and chlamydia with a pelvic exam and 77 were not. The screened group was of mean age 16.4 years compared to the unscreened group at 15.9 years, while the former also reported more STD-related symptoms than the unscreened group at the time of the initial PPCC visit. 29% of the 91 were positive for gonorrhea, chlamydia, or both. The average delay from pregnancy diagnosis to first prenatal clinic visit was 35.7 days. 53 of the 168 women presenting for prenatal care (32%) had STDs. Patients originally screened at the PPCC for STD, however, had a significantly lower prevalence of infection than the previously unscreened group. 22 of the group screened at the initial PPCC were infected, 12 of whom had been previously negative. The authors stress that primary care providers are missing an important therapeutic opportunity in this adolescent population by failing to identify and treat STDs at the initial diagnosis of pregnancy. The initial diagnosis of pregnancy in adolescents should always include screening for STD, with suspected infections treated immediately rather than waiting for definitive test results. Patients should also be given information on how to protect themselves from acquiring new STDs during pregnancy.

Delay in obstetrical care in newly diagnosed teenage pregnancy.

A retrospective study of 42 adolescent patients diagnosed as being pregnant between June 1987 and February 1990 at the Downtown Health Center (DHC), an inner city pediatric primary care clinic, was conducted to determine whether patients referred to a hospital-based Teen Pregnancy Clinic (TPC) were seen within a reasonable period of time. The frequency of sexually transmitted diseases (STDs) was also determined when these women were initially seen at TPC. Only 5 of the 42 patients seen at DHC had a pelvic exam prior to referral. Of the 40 patients seen at TPC, 20% were not seen until four weeks or more after initial diagnosis. Fifty percent had a STD. Pediatricians should recognize that pregnant teenagers may have a significant delay between diagnosis of pregnancy and entry into obstetrical care. Pelvic exam including cultures for STDs is recommended prior to referral.

Pancreatic cystic neoplasms.

Cystic neoplasms of the pancreas are relatively rare. This makes the evaluation and treatment of these tumors widely varied. The authors describe a patient who came to our hospital with complaints of abdominal pain, but no other related symptoms. Diagnostic evaluation of the patient yielded normal results, except for inspection and palpation of the abdominal areas, which revealed a large epigastric mass; this finding was confirmed subsequently by ultrasonographic examination and computed tomographic scanning. This article presents the case and reviews the literature, specifically related to diagnosis and current treatments.

Pretreatment metabotype as a predictor of response to sertraline or placebo in depressed outpatients: a proof of concept.

The purpose of this study was to determine whether the baseline metabolic profile (that is, metabotype) of a patient with major depressive disorder (MDD) would define how an individual will respond to treatment. Outpatients with MDD were randomly assigned to sertraline (up to 150 mg per day) (N=43) or placebo (N=46) in a double-blind 4-week trial. Baseline serum samples were profiled using the liquid chromatography electrochemical array; the output was digitized to create a 'digital map' of the entire measurable response for a particular sample. Response was defined as ≥50% reduction baseline to week 4 in the 17-item Hamilton Rating Scale for Depression total score. Models were built using the one-out method for cross-validation. Multivariate analyses showed that metabolic profiles partially separated responders and non-responders to sertraline or to placebo. For the sertraline models, the overall correct classification rate was 81% whereas it was 72% for the placebo models. Several pathways were implicated in separation of responders and non-responders on sertraline and on placebo including phenylalanine, tryptophan, purine and tocopherol. Dihydroxyphenylacetic acid, tocopherols and serotonin were common metabolites in separating responders and non-responders to both drug and placebo. Pretreatment metabotypes may predict which depressed patients will respond to acute treatment (4 weeks) with sertraline or placebo. Some pathways were informative for both treatments whereas other pathways were unique in predicting response to either sertraline or placebo. Metabolomics may inform the biochemical basis for the early efficacy of sertraline.