Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1.

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-ß interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-ß pathway and implicate this mechanism in the maintenance of genome integrity.

Automatic real-time three-dimensional cell tracking by fluorescence microscopy.

Live cell imaging has become an indispensable technique for cell biologists. However, when grown on coverslip glass used for live cell imaging many cultured cells move even during relatively short observation times and focus can drift as a result of mechanical instabilities and/or temperature fluctuations. Time-lapse imaging therefore requires constant adjustment of the imaging field and focus position to keep the cell of interest centred in the imaged volume. We show here that this limitation can be overcome by tracking cells in a fully automated way using the mass centre of cellular fluorescence. Combined with automated multiple location revisiting, this method dramatically increases the throughput of high-resolution live cell imaging experiments.

Smoking and Parkinson's disease in twins.

OBJECTIVE: To test the hypothesis that cigarette smoking protects against the development of PD. BACKGROUND: Smoking has been inversely associated with PD in many studies, but whether this reflects a biologic effect on the underlying disease process or merely confounding or selection bias remains uncertain. METHODS: The authors compared smoking histories in male twin pairs identified from the National Academy of Sciences--National Research Council World War II Veteran Twins Cohort. The amount of cigarettes smoked (in pack-years) was collected until the time of PD onset in the affected twin or until the time of death for the unaffected twin, whichever came first. Differences in pack-years smoked until PD onset and until 10 and 20 years before onset were compared using paired t-tests. Comparisons were made overall and stratified by zygosity and concordance for PD. To assess the role of shared environment, correlation for smoking behaviors was compared between pairs concordant and discordant for PD. RESULTS: Detailed smoking histories were available for 113 twin pairs in which at least one twin had PD (discordant pairs: 43 monozygotic [MZ], 50 dizygotic [DZ]; concordant pairs: 10 MZ, 10 DZ). Within-pair correlation for ever smoking was high in MZ pairs (phi = 0.47, p = 0.001) but not in DZ pairs (phi = 0.007, p = 0.96). In 33 discordant MZ pairs and 39 discordant DZ pairs in which at least one twin had smoked, the twins without PD smoked more than their brothers smoked (32.5 vs. 22.7 pack-years, p = 0.026). This was more marked in the MZ pairs (37.1 vs. 25.3 pack-years, p = 0.077) than in the DZ pairs (28.6 vs. 20.5 pack-years, p = 0.17). A similar relationship was seen when smoking dose was calculated only until 10 years before PD onset, suggesting that the lower dose of smoking in the twin with PD was not the result of early, undiagnosed disease. CONCLUSION: Within twin pairs, risk of PD is inversely correlated with the dose (in pack-years) of cigarette smoking. This effect is most pronounced in MZ twins, despite the high correlation for smoking. Because MZ twins are genetically identical and are similar behaviorally, this difference is unlikely to result from either genetic factors or environmental confounders. These results are compatible with a true biologic protective effect of cigarette smoking.

Serologic response to hepatitis B vaccine in HIV infected and high-risk HIV uninfected adolescents in the REACH cohort. Reaching for Excellence in Adolescent Care and Health.

PURPOSE: To evaluate hepatitis B (HBV) vaccine response rates in HIV infected and high-risk HIV uninfected youth and examine associations with responsiveness in the HIV infected group. METHODS: Cohorts within the Reaching for Excellence in Adolescent Care and Health (REACH) study population were defined based on receipt of HBV vaccine both retrospectively and prospectively. Sero-responsiveness was determined by HBsAb measurements. Testing was done for HBsAg, HBsAb, and HBcAb. For HBsAb, a value of > 10 International Units per liter was considered a positive response, and the data were collected as either positive or negative from each of the reporting laboratories. Covariates of responsiveness were explored in univariate and multivariate models for each cohort. RESULTS: Sixty-one subjects had received a three-dose vaccination course at the time of entry into REACH. HIV uninfected subjects had significantly higher rates of response by serology compared with HIV infected subjects (70% vs. 41.1%; chi(2) = .05; RR = .586, 95% CI: .36-.96). By the time of an annual visit 43 subjects had received three vaccinations with at least one occurring in the study period. The rates of response were similar for the HIV infected and uninfected groups (37.1% vs. 37.5%) in this cohort. Univariate and multivariate analysis in the prospective HIV infected group (N = 35) found an association between elevated CD8(+)/CD38(+)/HLA-DR(+) T cells and lack of HBV vaccine responsiveness (6.7% vs. 60%; chi(2) = .03; RR = .12, 95% CI: .02- .55). CONCLUSIONS: The poor HBV vaccine response rate in the HIV uninfected high-risk adolescents was unexpected and suggests that HBV vaccination doses have not been optimized for older adolescents. This is the first report of decreased responsiveness in HIV infected subjects being associated with elevated CD8(+)/CD38(+)/HLA(-)DR(+) T cells and suggests that ongoing viral replication and concomitant immune system activation decreases the ability of the immune system in HIV infected subjects to respond to vaccination.

Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells.

Live-cell imaging technology using fluorescent proteins (green fluorescent protein and its homologues) has revolutionized the study of cellular dynamics. But tools that can quantitatively analyse complex spatiotemporal processes in live cells remain lacking. Here we describe a new technique--fast multi-colour four-dimensional imaging combined with automated and quantitative time-space reconstruction--to fill this gap. As a proof of principle, we apply this method to study the re-formation of the nuclear envelope in live cells. Four-dimensional imaging of three spectrally distinct fluorescent proteins is used to simultaneously visualize three different cellular compartments at high speed and with high spatial resolution. The highly complex data, comprising several thousand images from a single cell, were quantitatively reconstructed in time-space by software developed in-house. This analysis reveals quantitative and qualitative insights into the highly ordered topology of nuclear envelope formation, in correlation with chromatin expansion - results that would have been impossible to achieve by manual inspection alone. Our new technique will greatly facilitate study of the highly ordered dynamic architecture of eukaryotic cells.

An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

Nucleocytoplasmic transport: diffusion channel or phase transition?

How exactly large molecules translocate through nuclear pores has been mysterious for a long time. Recent kinetic measurements of transport rates through the pore have led to a novel translocation model that elegantly combines selectivity with very high transport rates.

Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

Performance of antigens used in detecting delayed-type hypersensitivity in adolescents infected with the human immunodeficiency virus.

We examined the performance of delayed-type hypersensitivity (DTH) antigens employing a new Candida albicans product in a human immunodeficiency virus (HIV)-infected and nonanergic adolescent population. Diameters of induration (in millimeters) for three intradermally applied antigens (C. albicans, tetanus toxoid, and mumps) were compared in a population of HIV-infected 12 to 18 year olds at study entry in a national multicenter study of HIV disease progression. CD4+ T-cell counts were measured in quality-controlled laboratories. The influence of past immunization, gender, and clinical status on antigen reactivity was evaluated with contingency table comparisons and relative risk estimation. Nearly one-half of the 123 eligible subjects were untreated, and almost three-quarters were early in HIV disease by clinical indicators. There was no statistically significant difference in reactivity by past immunization status. Candida antigen (CASTA; Greer Laboratories) evoked DTH response in a significantly higher number of males and females at every level of induration (largest P value, 0.049 for male comparisons; all P values, <0.001 for females) and in subjects with early and intermediate HIV disease at every level of induration (all P values, <0.0001) than either tetanus or mumps antigens. No two-antigen combination was as useful as all three antigens across either gender or clinical categories, although candida and tetanus was the most useful two-antigen combination at indurations of <3 mm. The superior performance of a new C. albicans antigen may extend the utility of DTH assessment in monitoring immune function.

A new model for nuclear envelope breakdown.

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

The closeout process for a clinical trial terminated early for lagging enrollment and inadequate follow-up.

Closeout of a clinical trial, whether carried out to its original completion of full accrual and attendant follow-up or stopped prematurely because of early indications of efficacy or adverse toxicity, presents challenges in many areas. Closing a clinical trial that fails to adequately accrue and/or successfully follow up patients may exacerbate these problems. The issues involved in the early termination of the Low-Dose Oral Alpha Interferon Trial are described. Control Clin Trials 2001;22:49-55

The prevalence of anergy in human immunodeficiency virus-infected adolescents and the association of delayed-type hypersensitivity with subject characteristics.

PURPOSE: To examine the prevalence of anergy in HIV-infected adolescents and factors associated with its occurrence. METHODS: Anergy was defined as less than 2mm induration to each of three intradermally applied antigens (Candida albicans, tetanus toxoid, and mumps) between 24 and 96 hours in a population of HIV-infected adolescents aged 12-18 at entry in a national multicenter study of HIV disease progression. CD4(+) T-cell counts and plasma HIV-1 RNA were measured in quality controlled laboratories. Factors associated with the probability of anergy were examined with contingency table comparisons, tree-structured classification, and logistic regression analyses. RESULTS: Overall prevalence of anergy in this clinic-based population of 167 was 11% [7% in males and 12% in females (p = 0.57)]. The sole significant predictor of anergy was decreased CD4(+) T-cell count (p = 0.005). CONCLUSION: The prevalence of anergy is low in this HIV-infected population compared to older infected cohorts. The occurrence of differential rates of anergy in particular age and sex groupings that may be related to intrinsic immunologic differences requires further study.

A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition.

We describe a novel nuclear factor called mitotic chromosome-associated protein (MCAP), which belongs to the poorly understood BET subgroup of the bromodomain superfamily. Expression of the 200-kDa MCAP was linked to cell division, as it was induced by growth stimulation and repressed by growth inhibition. The most notable feature of MCAP was its association with chromosomes during mitosis, observed at a time when the majority of nuclear regulatory factors were released into the cytoplasm, coinciding with global cessation of transcription. Indicative of its predominant interaction with euchromatin, MCAP localized on mitotic chromosomes with exquisite specificity: (i) MCAP-chromosome association became evident subsequent to the initiation of histone H3 phosphorylation and early chromosomal condensation; and (ii) MCAP was absent from centromeres, the sites of heterochromatin. Supporting a role for MCAP in G(2)/M transition, microinjection of anti-MCAP antibody into HeLa cell nuclei completely inhibited the entry into mitosis, without abrogating the ongoing DNA replication. These results suggest that MCAP plays a role in a process governing chromosomal dynamics during mitosis.

Relationship of CD4+ T cell counts and HIV type 1 viral loads in untreated, infected adolescents. Adolescent Medicine HIV/AIDS Research Network.

The REACH Project (Reaching for Excellence in Adolescent Care and Health) of the Adolescent Medicine HIV/AIDS Research Network was designed as a study of an adolescent cohort composed of HIV-1-infected and -uninfected subjects. The goal of the analysis presented was to examine the relationship of CD4+ T cell counts and HIV-1 plasma viral loads in adolescents. The CD4+ T cell counts of 84 HIV+ subjects who were 13 to 19 years of age were measured at the clinical sites, using ACTG standardized techniques. HIV-1 viral loads in frozen plasma were determined by the NASBA/NucliSens assay at a central laboratory. Past and current treatment with antiretroviral drugs was determined by medical record abstraction and interview data. The slope of the line generated by regressing log10 HIV-1 RNA (copies/ml) versus CD4+ T cell counts of REACH subjects who are antiretroviral drug naive was negative and significantly different than zero. A negative association has also been reported for antiretroviral drug-naive, adult males in the Pittsburgh Men's Study, a component of MACS (Pitt-MACS) (Mellors J, et al.: Science 1996;272:1167). These data show that in adolescents, as in adults, HIV-1 RNA concentrations are correlated with corresponding absolute CD4+ T cell count. The slopes of the lines generated with data from each cohort were different (p = 0.003). In addition to age, there are sex and racial differences in the makeup of the two cohorts. Any or all of these differences may affect the slopes of the lines.

Dynamics and retention of misfolded proteins in native ER membranes.

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40 degrees C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.

Cytokine profile in genital tract secretions from female adolescents: impact of human immunodeficiency virus, human papillomavirus, and other sexually transmitted pathogens.

Quantitative enzyme-linked immunosorbent assays were used to measure interleukin (IL)-2, IL-10, and IL-12 in cervical secretions from female adolescents with and without sexually transmitted infections. Compared with human immunodeficiency virus [HIV]-negative patients, HIV-positive patients had higher concentrations of IL-10 (118.2 pg/mL vs. 34.5 pg/mL; P=.002) and IL-12 (175.5 pg/mL vs. 85.1; P=.03). IL-2 concentrations were not statistically different. Furthermore, genital tract infections were predictors of IL-10 and IL-12 concentrations. Coinfection with HIV and human papillomavirus predicted the highest IL-10 concentrations; coinfection with HIV, human papillomavirus, and other sexually transmitted pathogens predicted the highest IL-12 concentrations. The data indicate that concomitant infection of the genital tract with HIV and other viral, bacterial, or protozoan pathogens influences the local concentrations of some immunoregulatory cytokines.

Prevalence of and risks for cervical human papillomavirus infection and squamous intraepithelial lesions in adolescent girls: impact of infection with human immunodeficiency virus.

CONTEXT: Data suggest that in adults, human papillomavirus (HPV) infections and their sequalae, squamous intraepithelial lesions (SILs), occur more commonly among human immunodeficiency (HIV)-infected women because of the HIV-associated CD4+ T-cell immunosuppression. Since adolescents are more likely to be early in the course of HIV and HPV infections, the study of both infections in this age group may help elucidate their initial relationship. OBJECTIVE: To examine the prevalence of and risks for cervical HPV infection and SILs by HIV status in a population of adolescent girls. PARTICIPANTS: Subjects recruited at each of the 16 different US sites participating in a national study of HIV infection in adolescents. MAIN OUTCOME MEASURES: Cervical HPV DNA findings using polymerase chain reaction detection techniques and Papanicolaou smear from baseline visits. Infection with HPV was categorized into low- (rarely associated with cancer) and high- (commonly associated with cancers) risk types. RESULTS: Of 133 HIV-infected girls, 103 (77.4%) compared with 30 (54.5%) of 55 noninfected girls were positive for HPV (relative risk [RR], 1.4; 95% confidence interval [CI], 1.1-1.8). The risk was for high-risk (RR, 1.8; 95% CI, 1.2-2.7) but not low-risk (RR, 1.2; 95% Cl, 0.4-3.9) HPV types. Among the girls with HPV infection, 21 (70.0%) of the non-HIV-infected girls had normal cytologic findings compared with only 29 (29.9%) of the HIV-infected girls (P<.001). Multivariate analysis showed that HIV status was a significant risk for HPV infection (odds ratio [OR], 3.3; 95% CI, 1.6-6.7) and SIL (OR, 4.7; 95% CI, 1.8-14.8), but CD4 cell count and viral load were not associated with infection or squamous intraepithelial lesions. Only 9 girls had a CD4+ T-cell count of less than 0.2 cell X 10(9)/L. CONCLUSIONS: High prevalence of HPV infection in both groups underscores the risky sexual behavior in this adolescent cohort. Rates of HPV infection and SILs were higher among HIV-infected girls, despite similar sexual risk behaviors and the relatively healthy state of our HIV-infected group. Infection with HIV may enhance HPV proliferation through mechanisms other than CD4 immunosuppression, particularly early in the course of HIV infection.

Golgi membranes are absorbed into and reemerge from the ER during mitosis.

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.

Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras.

Understanding how membrane proteins are targeted to and retained within the nuclear envelope (NE) and the fate of these proteins during NE disassembly/reassembly in mitosis is central for insight into the function of the NE in nuclear organization and dynamics. To address these issues we have attached green fluorescent protein (GFP) to a well-characterized protein of the inner nuclear membrane, lamin B receptor, believed to be one of the major chromatin docking protein in the NE. We have used this construct in a variety of applications, including dual-color GFP time-lapse imaging, to investigate the mechanisms underlying protein targeting to the NE and NE breakdown and reassembly during mitosis. In this review, we present a summary of the results from such studies and discuss the photobleaching and imaging methodology on which they were derived.

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein.

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.