Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins.

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.

Calcium-dependent homotypic cell-cell adhesion, mediated by molecules such as E-cadherin, guides the establishment of classical epithelial cell polarity and contributes to the control of migration, growth, and differentiation. These actions involve additional proteins, including alpha- and beta-catenin (or plakoglobin) and p120, as well as linkage to the cortical actin cytoskeleton. The molecular basis for these interactions and their hierarchy of interaction remain controversial. We demonstrate a direct interaction between F-actin and alpha (E)-catenin, an activity not shared by either the cytoplasmic domain of E-cadherin or beta-catenin. Sedimentation assays and direct visualization by transmission electron microscopy reveal that alpha 1(E)-catenin binds and bundles F-actin in vitro with micromolar affinity at a catenin/G-actin monomer ratio of approximately 1:7 (mol/mol). Recombinant human beta-catenin can simultaneously bind to the alpha-catenin/actin complex but does not bind actin directly. Recombinant fragments encompassing the amino-terminal 228 residues of alpha 1(E)-catenin or the carboxyl-terminal 447 residues individually bind actin in cosedimentation assays with reduced affinity compared with the full-length protein, and neither fragment bundles actin. Except for similarities to vinculin, neither region contains sequences homologous to established actin-binding proteins. Collectively these data indicate that alpha 1 (E)-catenin is a novel actin-binding and -bundling protein and support a model in which alpha 1(E)-catenin is responsible for organizing and tethering actin filaments at the zones of E-cadherin-mediated cell-cell contact.

Surfactant protein a promotes attachment of Mycobacterium tuberculosis to alveolar macrophages during infection with human immunodeficiency virus.

The incidence of tuberculosis is increasing on a global scale, in part due to its strong association with human immunodeficiency virus (HIV) infection. Attachment of Mycobacterium tuberculosis to its host cell, the alveolar macrophage (AM), is an important early step in the pathogenesis of infection. Bronchoalveolar lavage of HIV-infected individuals demonstrated the presence of a factor which significantly enhances the attachment of tubercle bacilli to AMs 3-fold relative to a normal control population. This factor is surfactant protein A (SP-A). SP-A levels are increased in the lungs of HIV-infected individuals. SP-A levels and attachment of M. tuberculosis to AMs inversely correlate with peripheral blood CD4 lymphocyte counts. Elevated concentrations of SP-A during the progression of HIV infection may represent an important nonimmune risk factor for acquiring tuberculosis, even before significant depletion of CD4 lymphocytes in the peripheral blood occurs.

Taking The 'Sport' Out of Sports Parenting: Toward A Theory of Sport Related Parent-Child Communication Competency

Research has shown that over-emphasis on winning is the number one reason why approximately seventy percent of the forty million children who participate in youth sports will quit by age 13. This study utilized a constructivist grounded theory approach to investigate the role of parent-child communication within the context of youth sports. A total of 22 athletes and 20 parents were recruited through a Western university to discuss messages exchanged during youth sport participation. The results suggest that the delineation between messages of support and pressure is largely dependent on discursive work done by both parent and child. Parents who employed competent communicative strategies to avoid miscommunications regarding participation and sports goals were able to provide support and strengthen the relationship despite pressurized situations. The present study frames the youth sport dilemma within a developing conceptualization of communicative (in)competence and offers theoretical implications for sport related parent-child communication competency (SRPCCC).

Parenting influences on depression: A moderated mediated model

Little is known about the developmental processes through which parenting factors may influence clinical depression among youth. This study investigated whether parenting influences the onset of clinical depression through the mediating mechanism of negative attributional style, particularly under conditions of high stress, in a community sample of children and adolescents (N = 289). Results supported a moderated mediation model in which low levels of observed parent positive regard and sensitivity to distress during a youth stressor task were indirectly associated with an increased likelihood of experiencing an episode of depression over an18 month period, through the mediating influence of youth negative attributional style, but only for youth who also experienced a high number of peer stressors. These findings elucidate mechanisms through which parenting may contribute to risk for depression during the transition into and across adolescence.

The Clinical Utility of the Adult Attachment Projective Picture System: A Clinician's Perspective

The Adult Attachment Projective Picture System (AAP) is the first performance- based measure of adult attachment to be developed. The purpose of the measure is to provide a clinical understanding of an adult client's attachment status and associated coping mechanisms. The AAP is a relatively new measure that has yet to be examined from a utility perspective. In the current study, seven psychologists completed a structured survey in order to identify their perspectives of the AAP and its utility as a clinical instrument. A phenomenological qualitative analysis of the data was conducted to derive themes about the AAP and its clinical utility. Analyses aimed to answer the following: What clinical considerations do clinician's focus on when deciding to use this measure? What are common factors among clinician's who do use the measure as well as those who do not? What aspects of the measure are user-friendly and what aspects are difficult? General themes that emerged include (a) the clinical information provided by the AAP is viewed by those who use it as unique and beneficial; (b) time commitment and cost for the clinician are common considerations when clinician's are deciding whether or not to use the AAP or when pursuing training; (c) the AAP provides an increased understanding of one's relational capacities and defenses; and (d) the coding system and transcription process are difficult aspects of the AAP and influence how and/or when it is used. In addition to these themes, multiple respondents discussed potential changes for the AAP that would increase their future use of the instrument. Finally, the implications of these results are discussed.

The Trail of No Tears: Traditional Masculinity Ideologies and The Cycle of Dismissing-Avoidant Attachment

Raising boys in accordance with traditional masculinity ideologies is creating a mental health crisis among men. Socialization in accordance with traditional male gender roles causes boys to develop dismissing-avoidant attachments with their primary caregivers. Approaching subsequent relationships with a dismissing-attachment style creates disconnection between men and male peers, female partners, and their children. Many researchers advocate clinical interventions that perpetuate men's traditional fears of intimacy, however attachment theory provides an alternative lens through which clinicians may approach therapy with men. By engaging men in therapeutic attachment relationships, clinicians can inspire implicit and explicit learning of new attachment patterns. This experience by nature challenges traditional definitions of masculinity, and men may develop more congruent, adaptive, and healthy definitions of masculinity.

A Qualitative Study on Successful African American Marriages on How They Create and Repair Attachment Injuries Using Emotional Skillfulness.

The purpose of this phenomenological study was to describe the meanings that Ossie Davis and Ruby Dee Davis attribute to how they have adapted to their marriage and overcame conflict over the years. A purposive sample of this well-known African American married couple, who self-identified as being in a long-term, successful marriage, was used. The subjects of study were married for 56 years. An analysis using Colaizzi's (1978) method revealed 2 themes, with 13 and 2 subthemes respectively. The themes that emerged from the analysis of the formulated meanings were: (1) secrets to a successful marriage; and (2) sources of conflict in marriage. Secrets to a successful marriage included 13 subthemes: (a) egalitarian roles; (b) commitment; (c) forgiveness; (d) communication; (e) love; (f) honesty; (g) understanding the struggles of Black males and females; (h) friendship; (i) religion/ support from God; (j) compromise; (k) beliefs that marriage is a process; (l) emotional availability of spouse; and (m) feelings of security. In addition, the theme sources of conflict in marriage had two subthemes: (a) different decision making styles; and (b) experiences of abandonment. These findings provided insight from this couple's perspective on the secrets to a successful marriage and the ways in which they managed to make their marriage work, in light of the unique challenges that face African American marriages.

Adult Attachment Style on the MMPI-2: Does an Elevated Scale 4 Predict Dismissing Attachment?

This study examines some aspects of Caldwell's (200 I) hypotheses about the adaptations associated with elevations on the clinical scales of the Minnesota Multiphasic Personality Assessment-2 (MMPI-2). The argument is made that the adaptation to early rejection and neglect, which he attributes to Scale 4, parallels the adaptation represented by a dismissing attachment. Archival data of 72 individuals who completed assessment batteries that included both the MMPI-2 and that Adult Attachment Projective (AAP) were analyzed to determine if there was a relationship between elevations on Scale 4 and classification as dismissing attachment. No significant relationship was found for the primary hypothesis; however, some small effects were noted in related exploratory analyses. Possible reasons for the lack of a significant result are discussed a well as directions for future research.

Writ for the attachment of goods or estate: action brought by Edward Hutchinson, Treasurer of Harvard College, 1731 June 5

Writ of attachment authorizing the Suffolk County Sheriff to seize £150 in money or property from John Orme, George Lawrence, and Samuel Pearce, all of Watertown, in response to action brought by Harvard College Treasurer Edward Hutchinson regarding the bond of John White. The case-specific information is handwritten onto a printed form.

Transnational parenting and the well-being of Angolan migrant parents in Europe

Studies on the transnational family highlight the emotional difficulties of migrant parents separated from their children through international migration. This article consists of a large-scale quantitative investigation into the insights of transnational family literature by examining the well-being of transnational parents compared with that of parents who live with their children in the destination country. Furthermore, through a survey of Angolan migrant parents in both the Netherlands and Portugal, we compare the contexts of two receiving country. Our study shows transnational parents are worse off than their non-transnational counterparts in terms of four measures of well-being – health, life satisfaction, happiness, and emotional well-being. Although studies on migrant well-being tend to focus exclusively on the characteristics of the receiving countries, our findings suggest that, to understand migrant parents' well-being, a transnational perspective should also consider the existence of children in the migrant sending country. Finally, comparing the same population in two countries revealed that the receiving country effects the way in which transnational parenting is associated with migrant well-being.