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Insulin protects islets from apoptosis via Pdx1 and specific changes in the human islet proteome.

Insulin is both a hormone regulating energy metabolism and a growth factor. We and others have shown that physiological doses of insulin initiate complex signals in primary human and mouse beta-cells, but the functional significance of insulin's effects on this cell type remains unclear. In the present study, the role of insulin in beta-cell apoptosis was examined. Exogenous insulin completely prevented apoptosis induced by serum withdrawal when given at picomolar or low nanomolar concentrations but not at higher concentrations, indicating that physiological concentrations of insulin are antiapoptotic and that insulin signaling is self-limiting in islets. Insulin treatment was associated with the nuclear localization of Pdx1 and the prosurvival effects of insulin were largely absent in islets 50% deficient in Pdx1, providing direct evidence that Pdx1 is a signaling target of insulin. Physiological levels of insulin did not increase Akt phosphorylation, and the protective effects of insulin were only partially altered in islets lacking 80% of normal Akt activity, suggesting the presence of additional insulin-regulated antiapoptotic pathways. Proteomic analysis of insulin-treated human islets revealed significant changes in multiple proteins. Bridge-1, a Pdx1-binding partner and regulator of beta-cell survival, was increased significantly at low insulin doses. Together, these data suggest that insulin can act as a master regulator of islet survival by regulating Pdx1.

Analysis of Variance↗

Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-alpha regulate transcriptional signaling and apoptosis.

Insulin-like growth factor-binding protein (IGFBP)-3 regulates apoptosis in an IGF-independent fashion and has been shown to localize to nuclei. We cloned the nuclear receptor retinoid X receptor-alpha(RXR-alpha) as an IGFBP-3 protein partner in a yeast two-hybrid screen. Multiple methodologies showed that IGFBP-3 and RXR-alpha bind each other within the nucleus. IGFBP-3-induced apoptosis was abolished in RXR-alpha-knockout cells. IGFBP-3 and RXR ligands were additive in inducing apoptosis in prostate cancer cells. IGFBP-3 enhanced RXR response element and inhibited RARE signaling. Thus, RXR-alpha-IGFBP-3 interaction leads to modulation of the transcriptional activity of RXR-alpha and is essential for mediating the effects of IGFBP-3 on apoptosis.

Amino Acid Sequence↗

Kinetics of protein-protein interactions of connexins: use of enzyme linked sorbent assays.

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.

Animals↗

US11 of herpes simplex virus type 1 interacts with HIPK2 and antagonizes HIPK2-induced cell growth arrest.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase of the subfamily of dual-specificity Yak1-related kinase proteins. HIPK2 was first described as a homeodomain-interacting protein kinase acting as a corepressor for homeodomain transcription factors. More recently, it was reported that HIPK2 plays a role in p53-mediated cellular apoptosis and could also participate in the regulation of the cell cycle. US11 protein of herpes simplex virus type 1 is a multifunctional protein involved in the regulation of several processes related to the survival of cells submitted to environmental stresses by mechanisms that are not fully elucidated. In an attempt to better understand the multiple functions of US11, we identified cellular binding partners of this protein by using the yeast two-hybrid system. We report that US11 interacts with HIPK2 through the PEST domain of HIPK2 and that this interaction occurs also in human cells. This interaction modifies the subcellular distribution of HIPK2 and protects the cell against the HIPK2-induced cell growth arrest.

Adaptor Proteins, Signal Transducing↗

Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilization.

The AP-1 transcription factor plays an essential role in cell proliferation and tumorigenesis. It was previously shown that the fra-1 gene product is upregulated by various oncogenes and is involved in the in vitro and in vivo transformation of thyroid cells. Here we show that the ras oncogene-dependent accumulation of Fra-1 is mediated by a positive feedback mechanism which requires both transcriptional autoregulation and posttranslational stabilization of the protein. The oncogene-dependent transcriptional activation involves the cooperation between both Raf-dependent and Raf-independent pathways and is mediated by an AP-1 site within the fra-1 first intron, which becomes stably occupied by a transcriptionally active Fra-1-containing complex in ras-transformed cells. The posttranslational stabilization results in a drastic increase in the Fra-1 half-life in ras-transformed cells and is totally dependent on the activity of the MEK/ERK phosphorylation pathway. The analysis of the Fra-1 transactivation potential shows that the protein is able to stimulate a heterologous promoter in a ras-dependent manner, but the transactivating activity requires the recruitment of a heterodimeric partner. These data show that the alteration of multiple regulatory mechanisms is required for the constitutive activation of Fra-1 as a nuclear target of ras transformation.

Animals↗

Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals.

Despite multiple sexual exposure to HIV-1 virus, some individuals remain HIV-1 seronegative (exposed seronegative, ESN). The mechanisms underlying this resistance remain still unclear, although a multifactorial pathogenesis can be hypothesised. Although several genetic factors have been related to HIV-1 resistance, the homozigosity for a mutation in CCR5 gene (the 32 bp deletion, i.e. CCR5-Delta32 allele) is presently considered the most relevant one. In the present study we analysed the genotype at CCR5 locus of 30 Italian ESN individuals (case group) who referred multiple unprotected heterosexual intercourse with HIV-1 seropositive partner(s), for at least two years. One hundred and twenty HIV-1 infected patients and 120 individuals representative of the general population were included as control groups. Twenty percent of ESN individuals had heterozygous CCR5-Delta 32 genotype, compared to 7.5% of HIV-1 seropositive and 10% of individuals from the general population, respectively. None of the analysed individuals had CCR5-Delta 32 homozygous genotype. Sequence analysis of the entire open reading frame of CCR5 was performed in all ESN subjects and no polymorphisms or mutations were identified. Moreover, we determined the distribution of C77G variant in CD45 gene, which has been previously related to HIV-1 infection susceptibility. The frequency of the C77G variant showed no significant difference between ESN subjects and the two control groups. In conclusion, our data show a significantly higher frequency of CCR5-Delta 32 heterozygous genotype (p = 0.04) among the Italian heterosexual ESN individuals compared to HIV-1 seropositive patients, suggesting a partial protective role of CCR5-Delta 32 heterozygosity in this cohort.

Journal Article↗

HUA ENHANCER3 reveals a role for a cyclin-dependent protein kinase in the specification of floral organ identity in Arabidopsis.

In plants, organs are generated post-embryonically from highly organized structures known as meristems. Cell division in the meristem is closely integrated with cell fate specification and organ formation. The presence of multiple cyclin-dependent kinases (CDKs) and their partner cyclins in plants and other multicellular organisms probably reflects the complexity of cell cycle regulation within developmental contexts. The Arabidopsis genome encodes at least eight CDKs and 30 cyclins. However, no mutants in any CDKs have been reported, and the function of the great majority of these genes in plant development is unknown. We show that HUA ENHANCER3 (HEN3), which encodes CDKE, a homolog of mammalian CDK8, is required for the specification of stamen and carpel identities and for the proper termination of stem cells in the floral meristem. Therefore, CDK8 plays a role in cell differentiation in a multicellular organism.

Amino Acid Sequence↗

Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis.

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5' terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3' NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3' terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3' NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3' hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.

Base Sequence↗

Laboratory evaluation of the patient with erectile dysfunction.

Erectile dysfunction (ED) as a clinical entity is a problem that more than 50% of men will face as they age, and it can adversely affect overall quality of life and impact sexual partners. Our understanding of the pathophysiology and the multiple risk factors that contribute to ED has led to successful treatments, both non- surgical and surgical, over the past two decades. Now, more nonurologists and primary care providers are seeing patients for their initial evaluation. It is imperative that they approach the workup and evaluation in a logical and efficient manner. After a thorough history and physical examination, followed by a relevant and systematic laboratory evaluation, most cases can be effectively identified and medical treatment can be initiated. However, patients who continue to have erectile difficulties or fail initial oral treatment are candidates for specialized evaluation of vascular, neurogenic, and hormonal dysfunction, which can lead to more specific treatment or possible surgical management.

Clinical Laboratory Techniques↗

Human immunodeficiency virus postexposure prophylaxis for adolescents and children.

Children and adolescents are at risk for human immunodeficiency virus (HIV) infection. Transmission occurs through perinatal exposures, injecting drug use, consensual and nonconsensual sex, needle-stick and sharp injuries, and possibly some unusual contacts. Youth engaging in high-risk sexual activities are especially endangered. Half of the estimated worldwide 5.3 million new HIV infections occur in adolescents and young adults aged 15 to 24. Of 20 000 known new adult and adolescent cases in the United States, 25% involve 13- to 21-year-olds. More than 1.4 million children worldwide (aged 15 and younger) are believed to be infected, and >1640 new cases are diagnosed daily. Of the 432 000 people reported to be living with HIV or acquired immunodeficiency syndrome (AIDS) in the United States, 5575 are children under 13. HIV postexposure prophylaxis (PEP) is a form of secondary HIV prevention that may reduce the incidence of HIV infections. HIV PEP is commonly conceived of as 2 types: occupational and nonoccupational. Occupational HIV PEP is an accepted form of therapy for health care workers exposed to HIV through their jobs. A landmark study of healthcare workers concluded that occupational HIV PEP may be efficacious. Well-established US national guidelines for occupational HIV PEP exist for this at-risk population. Nonoccupational HIV PEP includes all other forms of HIV PEP, such as that given after sexual assault and consensual sex, injecting drug use, and needle-stick and sharp injuries in non-health care persons. Pediatric HIV PEP is typically the nonoccupational type. The efficacy of nonoccupational HIV PEP is unknown. The presumed efficacy is based on a collection of animal and human data concerning occupational, perinatal, and nonoccupational exposures to HIV. In contrast to occupational HIV PEP, there are no national US guidelines for nonoccupational HIV PEP, and few recommendations are available for its use for adolescents and children. Regardless of this absence, there is encouraging evidence supporting the value of HIV PEP in its various forms in pediatrics. Although unproven, the presumed mechanism for HIV PEP comes from animal and human work suggesting that shortly after an exposure to HIV, a window period exists during which the viral load is small enough to be controlled by the body's immune system. Antiretroviral medications given during this period may help to diminish or end viral replication, thereby reducing the viral inoculum to a more potentially manageable target for the host's defenses. HIV PEP is accepted practice in the perinatal setting and for health care workers with occupational injuries. The medical literature supports prescribing HIV PEP after community needle-stick and sharp injuries and after sexual assault from sources known or likely to be HIV-infected. HIV PEP after consensual unprotected intercourse between HIV sero-opposite partners has had growing use in the adult population, and can probably be utilized for children and adolescents. There is less documented experience and support for HIV PEP after consensual unprotected intercourse between partners of unknown HIV status, after prolonged or multiple episodes of sexual abuse from an assailant of unknown HIV status, after bites, and after the sharing of personal hygiene items or exposure to wounds of HIV-infected individuals. There are no formal guidelines for HIV PEP in adolescents and children. A few groups have commented on its provision in pediatrics, and some preliminary studies have been released. Our article provides a discussion of the data available on HIV transmission and HIV PEP in pediatrics. In our article, we propose an HIV PEP approach for adolescents and children. We recommend a stratified regimen, based on the work of Gerberding and Katz and other authors, that attempts to match seroconversion risk with an appropriate number of medications, while taking into account adverse side-effects and the amount of information that is typically available upon initial presentation. Twice daily regimens should be used when possible, and may improve compliance. HIV PEP should be administered within 1 hour of exposure. We strongly recommend that physicians trained in this form of therapy review the indications for HIV PEP within 72 hours of its provision. We advocate that due diligence in determining level of risk and appropriateness of drug selection be conducted as soon as possible after an exposure has occurred. When such information is not immediately available, we recommend the rapid treatment using the maximum level of care followed by careful investigation and reconsideration in follow-up or whenever possible. HIV PEP may be initiated provisionally after an exposure and then discontinued if the exposure source is confirmed to not be HIV-infected. In most cases, consultations with the experts in HIV care can occur after the rapid start of therapy. (ABSTRACT TRUNCATED)

Acquired Immunodeficiency Syndrome↗

Paternal influences on birthweight.

OBJECTIVE: To assess the influence of paternal size on birthweight after suitable control for maternal and fetal factors. DESIGN: Prospective observational study. SETTING: Delivery suite, City Hospital, Nottingham. SUBJECTS: 571 husbands/partners of unselected women delivering August 1992 to February 1993. MAIN OUTCOME METHODS: Individualised birthweight ratio and thereby an adjusted birthweight for a typical mother. The results of a multiple regression analysis with the individualised birthweight ratio as the dependent variable. RESULTS: When considered in isolation both paternal height and weight are significantly positively associated with crude and adjusted birthweight (p < 0.01, analysis of variance). Due to correlations of paternal size with maternal size and smoking habit, only paternal height is significant in the multiple regression analysis (p = 0.01). CONCLUSION: If the partner of an average woman is short (mean-2s.d.) then the baby will be 183 g lighter than if he is tall (mean + 2s.d.). This effect of paternal height on birthweight must be genetic and therefore should be taken into account when defining intra-uterine growth retardation and macrosomia.

Birth Weight↗

Requirements development for a patient computing system.

Critical parts of the software development life cycle are concerned with eliciting, understanding, and managing requirements. Though the literature on this subject dates back for several decades, practicing effective requirements development remains a current and challenging area. Some projects flourish with a requirements development process (RDP) that is implicit and informal, but this approach may be overly risky, particularly for large projects that involve multiple individuals, groups, and systems over time. At Partners HealthCare System in Boston, Massachusetts, we have applied a more formal approach for requirements development to the Patient Computing Project. The goal of the project is to create web-based software that connects patients electronically with their physician's offices and has the potential to improve care efficiency and quality. It is a large project, with over 500 function points. Like most technological innovation, the successful introduction of this system requires as much attention to understanding the business needs and workflow details as it does to technical design and implementation. This paper describes our RDP approach, and key business requirements discovered through this process. We believe that a formal RDP is essential, and that informatics as a field must include proficiencies in this area.

Boston↗

Fathers' experience of their partners' antepartum bed rest.

PURPOSE: To survey paternal worries, concerns, stresses, or problems and the type of support received by men whose partners were prescribed antepartum bed rest at home, or in the hospital, or both. DESIGN: Descriptive retrospective. A national subsample of 59 men whose mates were on pregnancy bed rest were randomly selected in 1995 from a nonrandom select sample of individuals who had contacted a bed-rest support group for information. METHODS: The Paternal Bed Rest Questionnaire of open-ended questions designed to detail paternal concerns, stresses, and supports was mailed to gathers. RESULTS: Major problems for fathers were assuming multiple roles, managing emotional responses, and caring for their partner. The major paternal worry was for the health of mate and fetus. Coping strategies included using tangible assistance; altering cognitive, behavioral, and emotional responses; and verbalizing worries. Fathers reported receiving little assistance from health care providers. CONCLUSIONS: Fathers experience extreme stress when pregnancy bed rest is prescribed for a mate. Family-centered care should include care of the partner whose mate is at high-risk. Interventions that reduce paternal worry and provide emotional and tangible support are needed.

Adaptation, Psychological↗

The multiple common point set problem and its application to molecule binding pattern detection.

Recognition of binding patterns common to a set of protein structures is important for recognition of function, prediction of binding, and drug design. We consider protein binding sites represented by a set of 3D points with assigned physico-chemical and geometrical properties important for protein-ligand interactions. We formulate the multiple binding site alignment problem as detection of the largest common set of such 3D points. We discuss the computational problem of multiple common point set detection and, particularly, the matching problem in K-partite-epsilon graphs, where K partitions are associated with K structures and edges are defined between epsilon-close points. We show that the K-partite-epsilon matching problem is NP-hard in the Euclidean space with dimension larger than one. Consequently, we show that the largest common point set problem between three point sets is NP-hard. On the practical side, we present a novel computational method, MultiBind, for recognition of binding patterns common to a set of protein structures. It performs a multiple alignment between protein binding sites in the absence of overall sequence, fold, or binding partner similarity. Despite the NP-hardness results, in our applications, we practically overcome the exponential number of multiple alignment combinations by applying an efficient branchand- bound filtering procedure. We show applications of MultiBind to several biological targets. The method recognizes patterns which are responsible for binding small molecules, such as estradiol, ATP/ANP, and transition state analogues.

Adenosine Triphosphate↗

Transcriptional coordination of the metabolic network in Arabidopsis.

Patterns of coexpression can reveal networks of functionally related genes and provide deeper understanding of processes requiring multiple gene products. We performed an analysis of coexpression networks for 1,330 genes from the AraCyc database of metabolic pathways in Arabidopsis (Arabidopsis thaliana). We found that genes associated with the same metabolic pathway are, on average, more highly coexpressed than genes from different pathways. Positively coexpressed genes within the same pathway tend to cluster close together in the pathway structure, while negatively correlated genes typically occupy more distant positions. The distribution of coexpression links per gene is highly skewed, with a small but significant number of genes having numerous coexpression partners but most having fewer than 10. Genes with multiple connections (hubs) tend to be single-copy genes, while genes with multiple paralogs are coexpressed with fewer genes, on average, than single-copy genes, suggesting that the network expands through gene duplication, followed by weakening of coexpression links involving duplicate nodes. Using a network-analysis algorithm based on coexpression with multiple pathway members (pathway-level coexpression), we identified and prioritized novel candidate pathway members, regulators, and cross pathway transcriptional control points for over 140 metabolic pathways. To facilitate exploration and analysis of the results, we provide a Web site (http://www.transvar.org/at_coexpress/analysis/web) listing analyzed pathways with links to regression and pathway-level coexpression results. These methods and results will aid in the prioritization of candidates for genetic analysis of metabolism in plants and contribute to the improvement of functional annotation of the Arabidopsis genome.

Arabidopsis↗

Building an academic-community partnered network for clinical services research: the Community Health Improvement Collaborative (CHIC).

OBJECTIVE: Community-based participatory research is recommended for research on health disparities and to improve uptake of clinical research findings. We describe the development of a multicenter consortium designed to support a community agency-academic partner infrastructure to support community-based, health-services research on multiple sources of health and healthcare disparities in local communities. DESIGN: We describe the development of the Los Angeles Community Health Improvement Collaborative (CHIC). RESULTS: The CHIC partners examined the research capacity and health priorities of its partners and developed a research agenda focused on four tracer conditions (depression, violence, diabetes, and obesity) and four areas for development of research capacity: public participation in all phases of research; understanding community and organizational context for clinical services interventions; practical clinical services trial methods; and advancing health information technology for clinical services research. The partners pooled resources to develop these areas for the tracer conditions. CONCLUSIONS: The challenges of a participatory approach to community-based clinical services research go beyond the significant methodologic and operational issues for specific projects and include building a sustainable capacity for research, community programs, and partnership across diverse communities and stakeholder organizations even when funding sources are not fully aligned with these goals.

Community Participation↗

Factors associated with risk for HIV infection among chronic mentally ill adults.

OBJECTIVE: Chronic mentally ill adults have been found to be at risk for HIV infection. The authors investigated the prevalence of HIV risk behaviors among psychiatric outpatients. Correlates of HIV risk factors and characteristics of patient relationships in which risk occurred were investigated. METHOD: Structured interviews were conducted with 95 chronic mentally ill adults from urban community support service programs. The interviews focused on sexual and substance use behavior, history of HIV risk behaviors, and relationship characteristics related to risk. RESULTS: The study showed that 27% of all patients had had two or more sex partners in the previous year and 18% had received money or drugs for sex. High rates of illicit drug use were also found, with frequent use of drugs or alcohol in association with sexual activity. Multiple regression analyses showed that use of illicit drugs, meeting sex partners in psychiatric clinics, and meeting partners in bars accounted for a substantial proportion of the variance in HIV risk behavior. CONCLUSIONS: These results suggest an urgent need for HIV prevention programs targeted at urban chronic mentally ill adults. Risk-producing characteristics of patient relationships and social networks should be addressed in the development of prevention interventions.

Adult↗

Characteristics of adolescents' sexual partners and their association with use of condoms and other contraceptive methods.

CONTEXT: While a number of studies have examined the association between individuals' characteristics and their contraceptive use, few studies have examined the influence of partners' characteristics on individuals' contraceptive use. METHODS: Using nationally representative data from the National Longitudinal Study of Adolescent Health, multiple logistic analyses were conducted to identify associations between the demographic characteristics of adolescents' heterosexual partners and adolescents' use of condoms or other contraceptive methods. RESULTS: The partners of white and black adolescents were likely to be similar to them, while the partners of Latino adolescents and of adolescents of "other" race or ethnicity were more likely to be of a different racial or ethnic group. Differences in age between adolescents and their partners were notable in all racial and ethnic groups. As adolescents age, the characteristics of their partners become more heterogeneous. The less similar adolescents and their partners are to one another--whether because of a difference in age, grade or school--the less likely adolescents are to use condoms and other contraceptive methods. CONCLUSIONS: Many adolescents have relationships with partners whose characteristics differ from theirs and with whom they are less likely to use condoms or other contraceptive methods. This behavior is more common as adolescents grow older. To provide appropriate counseling, sexuality educators and family planning providers need to consider the ways in which adolescents' relationships change as they age and discuss with them the dynamics of relationships involving partners who differ in age or other characteristics.

Adolescent↗