Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons.

Abeta-derived diffusible ligands (ADDLs) are abundant in AD brain, bind to hippocampal neurons and induce deficits in rodent cognition. To further investigate ADDL binding to neurons and identify antibodies that block this association, a panel of anti-Abeta and anti-ADDL antibodies was characterized for their ability to immuno-detect neuronally bound ADDLs and attenuate the binding of ADDLs to neurons. The results showed that anti-Abeta and anti-ADDL antibodies were able to abate ADDLs binding to hippocampal neurons, but to different degrees. Quantitative assessment of binding showed that one antibody, ACU-954 was markedly more effective at blocking ADDL binding than other antibodies assessed. ACU-954 was also found to block ADDL binding to hippocampal slice cultures, attenuate the ADDL-induced loss of dendritic spines and detect "natural ADDLs" in human AD tissue. These results demonstrated that antibodies that bind to and block ADDL binding to neurons can be identified, although their efficacy is conformationally specific since it is not readily apparent or predictable based on the core linear epitope or affinity for monomeric Abeta.

Comparison of storage phosphor computed radiography with conventional film-screen radiography in the recognition of pneumoconiosis.

Traditional film-screen radiography (FSR) has been useful in the recognition and evaluation of interstitial lung diseases, but is becoming increasingly obsolete. To evaluate the applicability of storage phosphor digital computed radiography (CR) images in the recognition of small lung opacities, we compared image quality and the profusion of small opacities between FSR and CR radiographs. We screened 1,388 working coal miners during the course of the study with FSR and CR images obtained on the same day from all participants. Each traditional chest film was independently interpreted by two of eight experienced readers using the International Labour Office (ILO) classification of radiographs of pneumoconiosis, as were CR images displayed on medical-grade computer monitors. The prevalence of small opacities (ILO category 1/0 or greater) did not differ between the two imaging modalities (5.2% for FSR and 4.8% for soft copy CR; p>0.50). Inter-reader agreement was also similar between FSR and CR. Significant differences between image modalities were observed in the shape of small opacities, and in the proportion of miners demonstrating high opacity profusion (category 2/1 and above). Our results indicate that, with appropriate attention to image acquisition and soft copy display, CR digital radiography can be equivalent to FSR in the identification of small interstitial lung opacities.

Rapidly progressive coal workers' pneumoconiosis in the United States: geographic clustering and other factors.

BACKGROUND: Despite significant progress made in reducing dust exposures in underground coal miners in the United States, severe cases of coal workers' pneumoconiosis (CWP), including progressive massive fibrosis (PMF), continue to occur among coal miners. AIMS: To identify US miners with rapidly progressive CWP and to describe their geographic distribution and associated risk factors. METHODS: Radiographic evidence of disease progression was evaluated for underground coal miners examined through US federal chest radiograph surveillance programmes from 1996 to 2002. A case of rapidly progressive CWP was defined as the development of PMF and/or an increase in small opacity profusion greater than one subcategory over five years. County based prevalences were derived for both CWP and rapidly progressive cases. RESULTS: A total of 886 cases of CWP were identified among 29 521 miners examined from 1996 to 2002. Among the subset of 783 miners with CWP for whom progression could be evaluated, 277 (35.4%) were cases of rapidly progressive CWP, including 41 with PMF. Miners with rapidly progressive CWP were younger than miners without rapid progression, were more likely to have worked in smaller mines (<50 employees), and also reported longer mean tenure in jobs involving work at the face of the mine (in contrast to other underground mining jobs), but did not differ with respect to mean underground tenure. There was a clear tendency for the proportion of cases of rapidly progressive CWP to be higher in eastern Kentucky, and western Virginia. CONCLUSIONS: Cases of rapidly progressive CWP can be regarded as sentinel health events, indicating inadequate prevention measures in specific regions. Such events should prompt investigations to identify causal factors and initiate appropriate additional measures to prevent further disease.

Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase.

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.

Discovery and analysis of inhibitors of the human immunodeficiency integrase.

An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.

The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates.

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.

The associations between immigrant status and risk-behavior patterns in Latino adolescents.

PURPOSE: The purpose of this study was to identify differences in patterns of risk-taking behavior among Latino adolescents with respect to immigrant status and in comparison to their native non-Hispanic white counterparts. METHODS: In fall of 1988 and spring of 1989 a confidential Teen Health Risk Survey was administered to 1,789 students at two high schools in northern California, both of which have a school health center on-site, and which had large numbers of Latino students enrolled. We divided the Latino adolescent population into two groups: Latinos who were born in the United States (native-born Latinos) and Latinos who had immigrated to the United States (Latino immigrants). Eight different risk-taking behaviors were identified for this study: alcohol, cigarette, marijuana, illicit drug use, self-violence, drunk driving, unintended pregnancy, and violence. RESULTS: We found that in general, Latino students engaged in a greater number of risk-taking behaviors than native non-Hispanic whites. The mean number of risk behaviors was highest for Latino immigrants (1.78), followed by native-born Latinos (1.71), and native non-Hispanic whites (.99). A t-test revealed that non-Hispanic whites were statistically different from both Latino populations in either level of risk-taking behavior (p < 0.05). CONCLUSIONS: This sample of immigrant Latino students appeared to be vulnerable to engaging in risk-taking behaviors and did not exhibit the anticipated cultural protective factors associated with recent immigrants. The extent and variety of risk-taking behaviors clearly call for the development of culturally sensitive and accessible health education and health care services within a broader array of social, educational and support services.

The impact of health insurance status on adolescents' utilization of school-based clinic services: implications for health care reform.

PURPOSE: 1) To examine variations among students with different health insurance coverage in their use of school-based clinics (SBCs), reasons for not receiving health care when needed, and reasons for using or not using SBCs, and 2) to determine if insurance status is a significant factor in predicting SBC use, after controlling for demographic variables and health status. METHODS: Confidential questionnaires were administered to 2,860 adolescents attending 3 urban high schools with on-site SBCs. Chi-square and multiple logistic regression analyses were used to assess differences among insurance groups in patterns of SBC use and reasons for clinic use/nonuse. RESULTS: Students with private insurance or HMO coverage had the highest rates of SBC utilization (67% & 66%) and students without health insurance and with Medicaid had the lowest (57% & 59%) (p < 0.01). While there was no difference among adolescents according to insurance group membership in their use of SBC medical services, a significantly higher proportion of students with Medicaid coverage used SBC mental health services. Students without health insurance were less likely to receive health care from any source when it was needed. After controlling for demographic variables and health status, no insurance factors remained significant. CONCLUSIONS: SBC users represent a variety of insurance groups. Health care reform efforts need to take into account the special needs of adolescents and the challenges they face in accessing care that go beyond financial barriers to care. SBC have been shown to provide a convenient and acceptable source of care, as well as offering the opportunity to provide preventive and primary care services to at-risk youth. As the country moves to a managed care environment potential partnerships with SBCs represent a unique opportunity to improve the delivery of care to adolescents, assuring increased access to a package of health services that they need.

Characteristics associated with contraceptive use among adolescent females in school-based family planning programs.

Among 162 young female family planning clients at four school-based health centers, a step-wise regression analysis shows that students' consistency of contraceptive use is associated with only a few specific service and provider characteristics. For example, clients who have more contacts with the family planning program use contraceptives more consistently than those with fewer contacts. On the other hand, young women whose follow-up visits are scheduled to occur within one month of their previous visit are less consistent contraceptive users than other clients. Contraceptive use is not related to whether contraceptives are dispensed on site, whether health education and counseling are provided by a health educator, whether contraceptive services are part of a comprehensive array of services that include medical or counseling services, or whether a family planning visit results in the dispensing of contraceptives or a prescription for contraceptives.

PIP: The study population included 162 adolescent female clients at 4 California school-based health clinics in secondary schools, who used reproductive services at least once during the 1990-91 school year. The total population of family planning clients during this period at the 4 sites was 744. Students (201) were selected who had at least 3 months of family planning visits between the first and the most recent visit. The profile included 80% females, 63% Hispanic females, 25% Black females, 7% Filipino females, 2% non-Hispanic White females, 1% Asian or Pacific Islander females, and 2% of other ethnicity. Contraceptive use was measured as a ratio of the number of months of contraceptive use at every act of intercourse to the number of months of involvement in family planning services. A month without sexual activity counted as continuous use. The range of use was 0-29 months. Linear and stepwise regression analyses were performed. The results showed no significant relationship between client characteristics and contraceptive use. A significant relationship was found, however, between the number of family planning visits and contraceptive use. The greater the number of family planning contacts, the higher the contraceptive use ratio. Each contact increased use by 3%. Less consistent contraceptive use was associated follow-up visits within a month of the previous visit. The contraceptive use ratio was not significantly related to availability of contraceptives on site, the type of health educator, the receipt of additional counseling or medical services, or dispensing contraceptives at each visit. Factors explaining contraceptive use were low at 7%. The inverse relationship at one-month follow-up may indicate that high-risk students are being identified, but the program is unable to convince student to maintain contraceptive use. Alternative approaches may be necessary to both identify high risk for contraceptive discontinuation users and to deal with poor contraceptive use. A study limitation was the selection process and the small number of subjects; the clinic's primary clients came for mental health or primary care visits. Student enrollment at the 4 schools was 9390 and almost 50% reported sexual activity, of which 33% reported frequent or consistent use of contraceptives.

Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase.

A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhibited a nonlinear dependence on integrase concentration, suggesting that the enzyme functions as a multimer. The oligomeric state was investigated by UV-photo-cross-linking of integrase-LTR oligonucleotide complexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine within the integrase recognition sequence. In the absence of divalent cation, integrase cross-linked to the LTR oligonucleotide as a single species whose mobility by SDS-polyacrylamide gel electrophoresis was consistent with the formation of tetramers. Using these techniques, analysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzyme are distinct, involving residues within the conserved "DD(35)E" and zinc finger motifs, respectively.