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PubMed2000

Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity.

UDP-glucose:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that operates as a gatekeeper for quality control by preventing transport of improperly folded glycoproteins out of the ER. We report the isolation of two cDNAs encoding human UDP-glucose:glycoprotein glucosyltransferase homologues. HUGT1 encodes a 1555 amino acid polypeptide that, upon cleavage of an N-terminal signal peptide, is predicted to produce a soluble 173 kDa protein with the ER retrieval signal REEL. HUGT2 encodes a 1516 amino acid polypeptide that also contains a signal peptide and the ER retrieval signal HDEL. HUGT1 shares 55% identity with HUGT2 and 31-45% identity with Drosophila, Caenorhabditis elegans, and Schizosaccharomyces pombe homologues, with most extensive conservation of residues in the carboxy-terminal fifth of the protein, the proposed catalytic domain. HUGT1 is expressed as multiple mRNA species that are induced to similar extents upon disruption of protein folding in the ER. In contrast, HUGT2 is transcribed as a single mRNA species that is not induced under similar conditions. HUGT1 and HUGT2 mRNAs are broadly expressed in multiple tissues and differ slightly in their tissue distribution. The HUGT1 and HUGT2 cDNAs were expressed by transient transfection in COS-1 monkey cells to obtain similar levels of protein localized to the ER. Extracts from HUGT1-transfected cells displayed a 27-fold increase in the transfer of [(14)C]glucose from UDP-[(14)C]glucose to denatured substrates. Despite its high degree of sequence identity with HUGT1, the expressed recombinant HUGT2 protein was not functional under the conditions optimized for HUGT1. Site-directed alanine mutagenesis within a highly conserved region of HUGT1 identified four residues that are essential for catalytic function.

Amino Acid Sequence↗
PubMedDate not supplied

Structure-based approaches to improve selectivity: CDK2-GSK3beta binding site analysis.

An evaluation and comparison of two different approaches, GRID/CPCA and GRIND/CPCA (CPCA = consensus principal component analysis; GRIND = GRid-INdependent Descriptors), suitable for visualizing the structural differences between related proteins is presented. Ten crystal structures of CDK2/cyclin A and GSK3beta solved in-house with different inhibitors were compared with the aim of highlighting regions that could be potential sites for gaining selectivity for CDK2 versus GSK3beta. The analyses pointed out remarkable differences in the backs of the CDK2-GSK3beta ATP binding pockets that guided the optimization toward a selective benzodipyrazole CDK2 inhibitor. The gain in selectivity can be associated with the two main differences in the ATP pocket between the enzymes. Phe80 of CDK2, the so-called gatekeeper residue often exploited for the design of kinase selective ligands, is replaced by a leucine in GSK3beta, and Ala144 is replaced by a cysteine. As a consequence of these mutations, CDK2 has a less elongated and less flat buried region at the back of the ATP pocket.

Adenosine Triphosphate↗
PubMed2001

Substrate recognition and selection by the initiation module PheATE of gramicidin S synthetase.

The initiation module of non-ribosomal peptide synthetases (NRPS) selects and activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizing L-phenylalanine (L-Phe) and activating it (by adenylation domain) as tightly bound L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), transferring it to the HS-phosphopantetheine arm of the holo-thiolation (holo-T) domain, and then epimerizing it (by epimerization domain) to the D-Phe-S-4'-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE adenylation domain with a number of proteinogenic amino acids and observed that three additional amino acids, L-Tyr, L-Trp, and L-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantetheine arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMPs and/or aminoacyl-S-4'-Ppant-acyl enzymes by the enzyme was not observed by three different assays for adenylation domain function. The microscopic reaction rates and thermodynamic equilibrium constants obtained from single-turnover studies of reactions of L-Phe, L-Trp, L-Tyr, and L-Leu with holoPheATE allowed us to construct free energy profiles for the reactions, revealing the kinetic and thermodynamic basis for substrate recognition and selection. In particular, the rates of epimerization of the L-aminoacyl-S-enzyme to the D-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300-, and 540-fold for L-Trp, L-Tyr, and L-Leu respectively, suggesting that the epimerization domain is an important gatekeeper for generation of the D-Phe-S-enzyme that starts gramicidin S chain growth.

Adenosine Monophosphate↗
PubMed2002

New insights into the mechanism of purple acid phosphatase through (1)H NMR spectroscopy of the recombinant human enzyme.

Proton NMR spectra of FeIII-FeII recombinant single polypeptide human PAP (recHPAP) have been measured at, above, and below its pH optimum, as have the spectra of inhibited forms containing fluoride and phosphate, analogues of the substrates hydroxide and phosphate esters, respectively. The results demonstrate that binding of inhibitory anions to the dinuclear mixed-valent site of recHPAP is controlled by protonation of a ligand to the dinuclear center. Thus, the group that is responsible for pKa,1 in the enzymatic activity versus pH profile functions as a "gatekeeper", whose protonation state controls anion binding to the mixed-valent dinuclear site. The correlation between the pKa values observed in kinetics studies and for the spectroscopic changes strongly suggests that this group is the nucleophilic hydroxide that attacks the phosphate ester substrate.

Acid Phosphatase↗
PubMed2025

Engineering Polyketide Stereocenters with Ketoreductase Domain Exchanges.

Polyketide synthases (PKSs) are versatile biosynthetic megasynthases capable of producing a diverse range of natural products with many applications, including in pharmaceuticals. The stereochemical precision of PKSs makes them a powerful tool for engineering tailored, unnatural polyketides; however, modifying the stereocenters of a PKS product while maintaining production levels remains a significant challenge. In this study, we systematically tested and evaluated strategies for ketoreductase (KR) domain exchanges, the domain responsible for setting stereocenters of polyketide products. After first optimizing the method for KR exchanges, we then performed 44 KR domain exchanges on three different PKSs to obtain high production of all four stereoisomers in vivo. By testing both one- and two-module PKS systems, we investigated how downstream modules process intermediates with altered stereochemistry and found that the configuration of the α-substituents was critical for gatekeeping by the ketosynthase (KS). To overcome this constraint, we investigated two different strategies for altering the KS domain, including introducing targeted mutations in the downstream KS, and exploring boundaries in exchanging the entire functional unit from the donor PKS. Both strategies successfully modified the KS stereocontrol with distinct trade-offs; the functional unit exchange resulted in higher titer improvements, though it was more likely to break the entire PKS. This study demonstrates a comprehensive approach to successfully engineering all four stereochemical configurations in multiple PKS systems, advancing our understanding of and ability to rationally modify polyketide stereochemistry through multiple engineering strategies.

Polyketides↗
PubMed2006

Structural insights into the ATP binding pocket of the anaplastic lymphoma kinase by site-directed mutagenesis, inhibitor binding analysis, and homology modeling.

Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors.

Adenosine Triphosphate↗
PubMed1999

Nucleic acid based techniques for the detection of rare cancer cells in clinical samples.

Solid tumors evolve from cells which have lost control functions safeguarding their genomic integrity by mutation within specific genes. Consequently, proliferating cells within a tumor differ slightly from generation to generation. This permits the continuous production of new and subsequent selection of the best adapted cells, which retain this capacity of self-evolution. The threat of neoplastic diseases is due to the clinical experience, that surgical resection of all cells with this potential for autonomous evolution is often not achievable, since they were spread to distant non-resectable sites in the organism before diagnosis and surgical removal of the primary tumor and might later grow out as metastatic lesion. Lack of diagnostic techniques to detect small preneoplastic lesions as well as single spread cancer cells often causes delayed diagnosis when curative therapy is no longer achievable. Recent advances in characterizing the molecular basis of genomic instability, identifying specific gatekeeper mutations and their functional consequences in neoplastic cells now permit the development of new highly sensitive tests to identify preneoplastic lesions as well as spread single cancer cells. These techniques carry a tremendous potential for simple cost effective cancer early detection and screening assays as well as for diagnostic applications to identify spread cancer cells. Thus, they most likely will soon guide indication for surgical and adjuvant therapy protocols for patients with preneoplastic or neoplastic lesions. However, due to the complexity of the assays and the great variety of technical aspects involved almost all diagnostic applications are not yet standardized. This poses significant problems for quality control as well as inter-laboratory comparability of the results and underlines the urgent demand for well controlled collaborative efforts to evaluate indications and diagnostic standards for these assays. Here, the theoretical background, basic principles and some diagnostic applications of these new tests are reviewed.

Animals↗
PubMed1999

Access to health care in the Scandinavian countries: ethical aspects.

The health care systems are fairly similar in the Scandinavian countries. The exact details vary, but in all three countries the system is almost exclusively publicly funded through taxation, and most (or all) hospitals are also publicly owned and managed. The countries also have a fairly strong primary care sector (even though it varies between the countries), with family physicians to various degrees acting as gatekeepers to specialist services. In Denmark most of the GP services are free. For the patient in Norway and Sweden there are out-of-pocket co-payments for GP consultations, with upper limits, but consultations for children are free. Hospital treatment is free in Denmark while the other countries use a system with out-of-pocket co-payment. There is a very strong public commitment to access to high quality health care for all. Solidarity and equality form the ideological basis for the Scandinavian welfare state. Means testing, for instance, has been widely rejected in the Scandinavian countries on the grounds that public services should not stigmatise any particular group. Solidarity also means devoting special consideration to the needs of those who have less chance than others of making their voices heard or exercising their rights. Issues of limited access are now, however, challenging the thinking about a health care system based on solidarity.

Delivery of Health Care↗
PubMed2001

When health means wealth, can bioethicists respond?

Around the world the wealthy can get their lives extended while the poor get little basic medical help. Over the same years that the field of bioethics has prospered and expanded, this disparity has increased. Reasons for the failure of bioethics to successfully address this health/wealth issue include its identification with the cognitive and social authority of medicine; its gatekeeping behavior; its funding sources; its questionable use of "principlism" and its emphasis on crises and dilemmas to the neglect of "housekeeping" issues. The work of most women in bioethics rarely addresses the health/wealth issue; if it does, their work may be ignored, as were the recommendations of Canadian feminists working under government grants. To achieve equity in health care, the structure of both medicine and bioethics needs to be changed. Yet, since bioethicists generally have accepted the status quo, this seems unlikely to happen.

Bioethics↗
PubMed1998

Why do patients prefer hospital emergency visits? A nested multinomial logit analysis for patient-initiated contacts.

This paper analyzes the nature of health care provider choice in the case of patient-initiated contacts, with special reference to a National Health Service setting, where monetary prices are zero and general practitioners act as gatekeepers to publicly financed specialized care. We focus our attention on the factors that may explain the continuously increasing use of hospital emergency visits as opposed to other provider alternatives. An extended version of a discrete choice model of demand for patient-initiated contacts is presented, allowing for individual and town residence size differences in perceived quality (preferences) between alternative providers and including travel and waiting time as non-monetary costs. Results of a nested multinomial logit model of provider choice are presented. Individual choice between alternatives considers, in a repeated nested structure, self-care, primary care, hospital and clinic emergency services. Welfare implications and income effects are analyzed by computing compensating variations, and by simulating the effects of user fees by levels of income. Results indicate that compensating variation per visit is higher than the direct marginal cost of emergency visits, and consequently, emergency visits do not appear as an inefficient alternative even for non-urgent conditions.

Choice Behavior↗
PubMed2004

Beyond the frontstage: trust, access, and the relational context in research with refugee communities.

This paper examines the role of trust and the concept of access as they affect the relational context in which community research, and research with refugee communities in particular, is conducted. Sociologist Irving Goffman's metaphor of frontstage and backstage behavior is used to illustrate the complexity and importance of developing of relations of trust, and thereby gaining "backstage" access, in communities that are generally closed to outsiders. It is argued that gaining authentic ("backstage") access to refugee communities, as well as other communities that have developed a self-protective insularity, is essential if we are to gather data that accurately reflect the actual beliefs, feelings, and experiences of community members. The paper then examines the puzzling lack of discussion regarding issues of trust, access, and the relational context in the literature on refugee mental health. Explanations for this apparent inattention are sought in the influence of the prevailing scientific paradigm that guides most research with refugees, and that shapes the values of the field's "gatekeepers," the editors and reviewers of scholarly journals. Throughout the paper, key points are illustrated with examples drawn from the author's research with refugees from Guatemala, Bosnia, and Afghanistan.

Developing Countries↗
PubMed2006

[GERD: endoscopic antireflux therapies].

A couple of minimally-invasive, endoscopic antireflux procedures have been developed during the last years. Beside endoscopic suturing these included injection/implantation technique of biopolymers and application of radiofrequency. Radiofrequency (Stretta) has proved only a very modest effect, while implantation techniques have been abandoned due to lack of long-term efficacy (Gatekeeper) or serious side effects (Enteryx). While first generation endoluminal suturing techniques (EndoCinch, ESD) demonstrated a proof of principle their lack of durability, due to suture loss, led to the development of a potentially durable transmural plication technique (Plicator). In a prospective-randomized, sham-controlled trial the Plicator procedure proved superiority concerning reflux symptoms, medication use and esophageal acid exposure (24-h-pH-metry). While long-term data have to be awaited to draw final conclusions, technical improvements will drive innovation in this field.

Cardia↗
PubMed2002

Pathways to care: help seeking behaviour in first episode psychosis.

OBJECTIVE: To determine the number of attempts it took before patients with a first episode of psychosis received adequate help, the signs or symptoms that led them to seek help and the people from whom they attempted to seek help. METHOD: Subjects were 86 individuals with a schizophrenia spectrum disorder, mainly schizophrenia, who were attending a comprehensive program for early psychosis treatment. RESULTS: Help-seeking attempts began in the prodromal phase of the illness and continued into the psychotic phase. Concerning behaviours ranged from more general symptoms to psychotic symptoms. A range of contacts were made early on but emergency services were most often the contact that helped individuals obtain appropriate treatment for psychosis. CONCLUSION: Improved public education and gatekeeper education might reduce the time required for individuals developing a psychosis to receive timely and adequate care.

Adolescent↗
PubMed1990

Use of inpatient mental health services by members of ethnic minority groups.

National data on psychiatric hospitalization point to marked ethnic-related differences. Blacks and Native Americans are considerably more likely than Whites to be hospitalized; Blacks are more likely than Whites to be admitted as schizophrenic and less likely to be diagnosed as having an affective disorder; Asian Americans/Pacific Islanders are less likely than Whites to be admitted, but remain for a lengthier stay, at least in state and county mental hospitals. These differences are clear-cut, but they ignore a major source of care: psychiatric hospitalization in placements other than psychiatric units and hospitals. Explanations for observed minority-White differences in hospitalization can be evaluated only partially or not at all: Such explanations include ethnic-related differences in socioeconomic standing and in the prevalence of major psychopathology; differential stigma, or capacity to tolerate or support a dysfunctional significant other; access and use of alternative services; and bias in the behavior of gatekeepers, especially practitioners assigning diagnostic labels and making involuntary commitment decisions. More research is needed to help explain these striking differences in utilization.

Diagnosis, Differential↗
PubMed2003

Family and legal indicators of child adjustment to divorce among families with young children.

This research used structural equation modeling to examine relations among family dynamics, attorney involvement, and the adjustment of young children (0-6 years) at the time of parental separation. The article presents baseline data (N = 102 nonresidential fathers and N = 110 primary caretaking mothers) from a larger longitudinal study. Results showed that the effects of parental conflict on child outcomes were mediated by paternal involvement, the parent-child relationship, and attorney involvement. A scale assessing parental gatekeeping yielded two significant factors: Spouse's Influence on Parenting and Positive View of Spouse. Paternal involvement was related to children's adaptive behavior, whereas negative changes in parent-child relationships predicted behavior problems. Mothers who experienced greater psychological symptomatology were less likely to utilize an attorney, which in turn predicted greater internalizing problems in their children.

Adolescent↗
PubMed2003

Family processes within three-generation households and adolescent mothers' satisfaction with father involvement.

This investigation used a family systems perspective to examine how family dynamics within 3-generation households were associated with mothers' satisfaction with father involvement. The participants were low-income African American adolescent mothers (n=148) and grandmothers recruited at delivery and followed over 6 months. The overall model explained 68% of the variance in satisfaction with father involvement. Fathers who were involved with caregiving activities had positive relationships with adolescent mothers and grandmothers. Grandmothers served as gatekeepers; when grandmothers reported positive relationships with the 2 young parents, adolescent mothers reported positive relationships with their male partners. Mothers who reported positive partner relationships also reported high parenting efficacy and satisfaction with father involvement. Efforts to increase paternal involvement should focus on role clarification for grandmothers and fathers and on parenting activities for mothers and fathers, regardless of their romantic relationship.

Adolescent↗
PubMed1999

Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

ATP Binding Cassette Transporter 1↗
PubMed2000

S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility.

Many flowering plants avoid inbreeding through a genetic mechanism termed self-incompatibility. An extremely polymorphic S-locus controls the gametophytic self-incompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the S-alleles present in the diploid style. The only known product of the S-locus is an S-RNase expressed in the mature style. The pollen component to this cell-cell recognition system is unknown and current models propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.

Pollen↗