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A strategic approach to the psychiatric workforce dilemma.

To help build consensus in the field of psychiatry about future psychiatric manpower needs, a 13-step strategic approach to the workforce issue is described. The steps include recognizing the importance of the assumptions that underlie workforce requirements; selecting credible and professional leadership with vision and courage; adopting a strategic plan to clarify workforce assumptions; re-examining the structure and function of established programs; preparing trainees for work in the 21st century; preserving psychiatry's humanistic tradition; enlisting the support of nonacademic psychiatrists; and reinforcing involvement in the fiscal and political aspects of medicine. They also include focusing attention on important policy issues; securing the support of patients, families, and advocates; endorsing a multidisciplinary, biopsychosocial approach to the evaluation and treatment of mental illness; minimizing divisive conflicts within and between national organizations; and developing strategic alliances with other medical disciplines. Implications of the 13-step approach are outlined for psychiatric clinicians, educators, and researchers, as well as for the organizations that serve them.

Education, Medical↗

Critical role of a lipoyl cofactor of the dihydrolipoyl acetyltransferase in the binding and enhanced function of the pyruvate dehydrogenase kinase.

Mammalian pyruvate dehydrogenase kinase binds to the lipoyl domain region of the core structure forming dihydrolipoyl acetyltransferase (E2) subunits. The bound kinase has a greatly enhanced rate in phosphorylating E2-bound pyruvate dehydrogenase (E1) tetramers versus the rate at which resolved kinase phosphorylates dissociated E1. This E2-activated kinase function was completely prevented by selective alkylation of reduced lipoyl groups while kinase and E1 binding to the E2 core were retained. Selective removal of lipoyl cofactors from intact E2 by treatment with Enterococcus faecalis lipoamidase decreased kinase activity by 4-fold and caused selective release of a major portion of the kinase from E2 in a sucrose-step gradient procedure. Selective and reversible modification of the lipoyl groups of E2 subunits also allowed the kinase to be dissociated under mildly chaotropic conditions. Thus, the lipoyl prosthetic group on one of the two lipoyl domains of E2 subunits is critically important for maintaining E2-activated kinase function and contributes to binding of the kinase to E2. Since removal of the lipoyl group weakened kinase binding to E2 more than modifying lipoyl thiols, it is suggested that the hydrophobic inner portion of the lipoyl conjugate (i.e., lysine carbons and C1 to C5 of the lipoic acid) is important in the binding of the kinase.

Acetylation↗

Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer.

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.

Clathrin↗

Combined conformational search and finite-difference Poisson-Boltzmann approach for flexible docking. Application to an operator mutation in the lambda repressor-operator complex.

The N-terminal domain of the phage lambda repressor binds as a dimer to its palindromic DNA operator sequence. In addition to a helix-turn-helix DNA recognition motif, the first six amino acids of the phage lambda repressor form a flexible peptide segment which wraps around DNA. Site-directed mutagenesis studies have shown that amino acid replacements or partial removal of the arm structure, or changes in the DNA sequence contacting the N-terminal arm, can lower the repressor-operator binding affinity by several orders of magnitude. The finite-difference Poisson-Boltzmann approach in combination with a conformational search procedure was used to study energetic contributions of the lambda arm to repressor-operator recognition based on the high resolution X-ray structure. It allows for the local relaxation of the structure upon changing the DNA sequence in the lambda arm binding region. A simplified potential energy function including torsional, truncated Lennard-Jones and approximate electrostatic terms is used in the initial step to screen out energetically unfavorable structures. The electrostatic energy of selected conformations is subsequently calculated more accurately using the finite-difference Poisson-Boltzmann approach. The method was applied to study the effect of a C-->T mutation at position 6 of the consensus half-site of the operator. This base-pair contacts Lys4 which is part of the arm segment. Keeping only the Lys4 side-chain mobile and with the wild-type DNA operator sequence, several conformations close to the X-ray structure were identified as those with lowest energy. In the case of the DNA mutation, lowest energy conformations differed significantly from those selected for the wild-type sequence. These initial calculations indicate that the approach might be a useful tool to estimate conformational and energetic effects upon mutagenesis of protein-DNA complexes.

Base Sequence↗

Increased transcomplementation properties of plasmids carrying HSV-1 origin of replication and packaging signals.

This report describes a very simple method that facilitates the analysis of the functional activity of essential genes of herpes simplex virus type 1 (HSV-1) in cell culture. The method, which depends on the use of complementing plasmids containing a virus origin of replication (ori s) and a packaging signal (amplicon plasmids), was tested for its ability to detect complementation of the defective HSV-1 Cgal delta 42 virus, a UL42 null mutant, by amplicon plasmids carrying either wild-type (pA-UL42) or mutated alleles (pA-delta UL42) of the HSV-1 UL42 gene. In nonpermissive Vero cells transfected with amplicon plasmid pA-UL42 and superinfected with the defective Cgal delta 42 virus, both the plasmid and the helper genomes were amplified and packaged, giving rise to a self-complementing amplicon/helper virus population able to disseminate and to form lytic plaques in cell culture. These plaques were due to complementation and not to recombination between the defective virus and the amplicon plasmid as confirmed by Southern blot analysis of individual plaques. No complementation was observed by superinfection of cells transfected with the noncomplementing pA-delta UL42 amplicon plasmid. Instead, small foci were observed in cells transfected with plasmids which express wild-type UL42 but were unable to amplify or to become packaged, and the few true lytic plaques that were observed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complementation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess function activity of an HSV-1 essential gene in a one-step experiment and (ii) oris-carrying plasmids could be instrumental in the production and selection of recombinant HSV-1 vectors.

Animals↗

Cloning, mapping and characterization of the human RAB27A gene.

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.

3' Untranslated Regions↗

Managing the centrosome numbers game: from chaos to stability in cancer cell division.

Aneuploid tumor cells can arise through multipolar mitosis caused by supernumerary centrosomes. Multipolar spindles, however, are antagonistic to cell viability. Thus, most cells derived from such an aberrant mitosis would be eliminated by apoptosis. A rare daughter cell, through chance acquisition of an appropriate chromosome complement and/or gene dosage, could survive and contribute to a clone of aneuploid tumor cells. Survival and perpetuation of the clone, however, requires an additional step - the resumption of mitotic stability through the assembly of a bipolar, not multipolar, spindle. Either selective inactivation of the extra centrosomes or their coalescence into two functional spindle poles corrects the problem of centrosome excess. Current data support coalescence as a mechanism for regulating the number of functional centrosomes in tumor cells.

Aneuploidy↗

Role of hyperglycaemia-related acidosis in ischaemic brain damage.

Although previous results have shown unequivocally that pre-ischaemic hyperglycaemia aggravates brain damage due to transient ischaemia, several questions have remained unanswered. First, is the effect of hyperglycaemia due to a further fall in intra- and extracellular pH? Second, is aggravation of damage a step function of a continuous function of plasma glucose concentration or of pH? Third, which are the mechanisms responsible for aggravation of damage, notably for the transformation of selective neuronal damage to infarction, for oedema development, and for post-ischaemic seizures? Recent results have provided new information on all of these issues. Thus, normoglycaemic animals with superimposed hypercapnia showed a similar, albeit not identical, aggravation of ischaemic damage, suggesting that acidosis is one major mediator. Furthermore, experiments with graded increase in plasma glucose concentration revealed a threshold effect at values of 10-12 mM, while microelectrode measurements showed a narrow extracellular pH range (6.4-6.5) for post-ischaemic seizure development. These results suggest that aggravation of damage due to excessive acidosis is due to mechanisms with a steep pH dependence. Finally, results are now at hand suggesting that the effect of acidosis is not mediated by a further perturbation of cell calcium metabolism. The more likely mediators are free radicals. Thus, acidosis is known to enhance iron-catalysed production of reactive oxygen species, probably by releasing iron from its bindings to transferrin, ferritin and other proteins.

Acidosis↗

Ras-GRF activates Ha-Ras, but not N-Ras or K-Ras 4B, protein in vivo.

Human cells contain four homologous Ras proteins, but it is unknown whether these homologues have different biological functions. As a first step in determining if Ras homologues might participate in distinct signaling cascades, we assessed whether a given Ras guanine nucleotide exchange factor could selectively activate a single Ras homologue in vivo. We found that Ras-GRF/Cdc25Mm activates Ha-Ras, but does not activate N-Ras or K-Ras 4B, protein in vivo. Moreover, our results suggested that residues within the C-terminal hypervariable domains of Ras proteins may dictate, at least in part, the specificity of Ras-GRF/CDC25Mm for Ha-Ras protein. Our studies represent the first biochemical evidence that a Ras GEF can selectively activate a single Ras homologue in vivo. Selective activation of a single Ras homologue by Ras-GRF/Cdc25Mm or other Ras guanine nucleotide exchange factors could potentially enable each of the Ras homologues to participate in different signal transduction pathways.

3T3 Cells↗

Large scale purification of factor X by hydrophobic chromatography.

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.

Chromatography↗

The application accuracy of stereotactic frames.

The purpose of incorporating stereotactic methodology into neurosurgical operations is to achieve a consistently high degree of accuracy in localizing intracranial targets. Therefore, the limits of resolution for the operation are a function of the accuracy of the particular stereotactic frame system. The total clinically relevant error (application accuracy) comprises errors associated with each procedural step, including imaging, target selection, vector calculations, and the mechanical errors of stereotactic frames. To evaluate these parameters, a systematic error analysis was carried out with four commonly used stereotactic devices: the Brown-Roberts-Wells, the Cosman-Roberts-Wells, the Kelly-Goerss COMPASS (modified Todd-Wells), and the Leksell frames. Over 21,500 independent accuracy test measurements were made with 11,000 computed tomograms. The results suggest a potentially significant degree of error in the application accuracy of all stereotactic instruments, which is accentuated by but not entirely due to imaging-associated errors. Clinically encountered levels of weightbearing by stereotactic frames may have a pronounced effect on their mechanical accuracy. Both the reapplication of aiming arc assemblies and the use of phantom base units introduce independent sources of mechanical inaccuracy into stereotactic procedures. The scope of individual error values and their determining factors must be considered with every clinical use of stereotactic frame systems.

Brain Diseases↗

Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.

The sympathetic nervous system controls lipolysis in fat by activation of four adrenergic receptors: beta1, beta2, beta3, and alpha2. During pregnancy, maternal metabolism presents anabolic and catabolic phases, characterized by modifications of fat responsiveness to catecholamines. The contributions of the four adrenergic receptors to adipocyte responsiveness during pregnancy have never been studied. Our aim was to evaluate the influence of pregnancy on adrenergic receptor-mediated lipolysis in rabbit white adipocytes. Functional studies were performed using subtype-selective and non-selective adrenergic receptor agonists. Overall adrenergic responsiveness was measured with the physiological agonist epinephrine. Non-adrenergic agents were used to evaluate different steps of the lipolytic cascade. The alpha2- and beta1/beta2-adrenergic receptor numbers were determined with selective radioligands. Non-adrenergic agents revealed that pregnancy induced an intracytoplasmic modification of the lipolytic cascade in inguinal but not in retroperitoneal adipocytes. Pregnancy induced an increase in beta1- and specially beta3-mediated lipolysis. The amounts of adipocyte beta1/beta2- and alpha2-adrenergic receptors were increased in pregnant rabbits. Epinephrine effects revealed an increased contribution of alpha2-adrenergic receptor-mediated antilipolysis in adipocytes from pregnant rabbits. These results indicate that pregnancy regulates adipocyte responsiveness to catecholamines mainly via the alpha2- and beta3-adrenergic pathways. Pregnancy induces an intracytoplasmic modification of the lipolytic cascade, probably via hormone-sensitive lipase, with differences according to fat location.-Bousquet-Mélou, A., C. Muñoz, J. Galitzky, M. Berlan, and M. Lafontan. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.

Adipocytes↗

Plant tagnology.

Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence-function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species.

Journal Article↗

New, simple approach for maximal pudendal nerve exposure: anomalies and prospects for functional reconstruction.

PURPOSE: Functional neosphincters after pudendal nerve anastomosis proved possible in animal models and may be applicable in humans, but access is a recognized problem. We report the occurrence of pudendal nerve anomalies, its implications for reconstruction, and describe a new approach for maximal exposure. METHODS: Adult human cadavers were positioned prone and dissected via a gluteal approach. Pudendal nerve variations and physical measurements were analyzed statistically. RESULTS: A new, simple, four-step approach (surface landmarks and exposure of gluteus maximus muscle, sacrotuberous ligament, and pudendal neurovascular bundle) permitted optimal pudendal nerve exposure in all 14 human cadavers (28 limbs). Six were males and had a mean age of 82 (range, 58-102) years. Two anomalies, Type 1 (2-trunked) and Type 2 (3-trunked), of the pudendal nerve were recognized in 30 percent of cadavers, with a left-to-right ratio of 2.5:1. Mean pudendal nerve length over the ischial spine was 23.9 (range, 19-28) mm right, 24.2 (range, 19-28) mm left (P = 0.54), but its diameter measured 5.2 mm (right) and 4.9 mm (left; P = 0.04). Mean length of pudendal nerve trunk exposed after reflection of the sacrotuberous ligament was 55 (range, 44-75) mm on either side before division into terminal branches. The number and percent frequency of inferior rectal nerve on both sides were 1 (13 percent), 2 (76 percent), and 3 (11 percent), respectively, with a mean length of 27.1 (range, 21-34) mm right and 27.9 (range, 20-33) mm left (P = 0.31). CONCLUSION: A simple four-step approach to the pudendal nerve contributes to improved access in all cases. It facilitates reconstruction because it allows accurate nerve selection and recognition of potential anomalies that might influence functional outcome.

Aged↗

Materials management information systems.

The hospital materials management function--ensuring that goods and services get from a source to an end user--encompasses many areas of the hospital and can significantly affect hospital costs. Performing this function in a manner that will keep costs down and ensure adequate cash flow requires effective management of a large amount of information from a variety of sources. To effectively coordinate such information, most hospitals have implemented some form of materials management information system (MMIS). These systems can be used to automate or facilitate functions such as purchasing, accounting, inventory management, and patient supply charges. In this study, we evaluated seven MMISs from seven vendors, focusing on the functional capabilities of each system and the quality of the service and support provided by the vendor. This Evaluation is intended to (1) assist hospitals purchasing an MMIS by educating materials managers about the capabilities, benefits, and limitations of MMISs and (2) educate clinical engineers and information system managers about the scope of materials management within a healthcare facility. Because software products cannot be evaluated in the same manner as most devices typically included in Health Devices Evaluations, our standard Evaluation protocol was not applicable for this technology. Instead, we based our ratings on our observations (e.g., during site visits), interviews we conducted with current users of each system, and information provided by the vendor (e.g., in response to a request for information [RFI]). We divided the Evaluation into the following sections: Section 1. Responsibilities and Information Requirements of Materials Management: Provides an overview of typical materials management functions and describes the capabilities, benefits, and limitations of MMISs. Also includes the supplementary article, "Inventory Cost and Reimbursement Issues" and the glossary, "Materials Management Terminology." Section 2. The MMIS Selection Process: Outlines steps to follow and describes factors to consider when selecting an MMIS. Also includes our Materials Management Process Evaluation and Needs Assessment Worksheet (which is also available online through ECRInet(TM)) and a list of suggested interview questions to be used when gathering user experience information for systems under consideration. Section 3A. MMIS Vendor Profiles: Presents information for the evaluated systems in a standardized, easy-to-compare format. Profiles include an Executive Summary describing our findings, a discussion of user comments, a listing of MMIS specifications, and information on the vendor's business background. Section 3B. Discussion of Vendor Profile Conclusions and Ratings: Presents our ratings and summarizes our rationale for all evaluated systems. Also includes a blank Vendor Profile Template to be used when gathering information on other vendors and systems. We found that, in general, all of the evaluated systems are able to meet most of the functional needs of a materials management department. However, we did uncover significant differences in the quality of service and support provided by each vendor, and our ratings reflect these differences: we rated two of the systems Acceptable--Preferred and four of the systems Acceptable. We have not yet rated the seventh system because our user experience information may not reflect the vendor's new ownership and management. When this vendor provides the references we requested, we will interview users and supply a rating. We caution readers against basing purchasing decisions solely on our ratings. Each hospital must consider the unique needs of its users and its overall strategic plans--a process that can be aided by using our Process Evaluation and Needs Assessment Worksheet. Our conclusions can then be used to narrow down the number of vendors under consideration...

Computer Security↗

The role of limb movements in maintaining upright stance: the "change-in-support" strategy.

Change-in-support strategies, involving stepping or grasping movements of the limbs, are prevalent reactions to instability and appear to play a more important functional role in maintaining upright stance than has generally been appreciated. Contrary to traditional views, change-in-support reactions are not just strategies of last resort, but are often initiated well before the center of mass is near the stability limits of the base of support. Furthermore, it appears that subjects, when given the option, will select these reactions in preference to the fixed-support "hip strategy" that has been purported to be of functional importance. The rapid speed of compensatory change-in-support reactions distinguishes them from "volitional" arm and leg movements. In addition, compensatory stepping reactions often lack the anticipatory control elements that are invariably present in non-compensatory stepping, such as gait initiation. Even when present, these anticipatory adjustments appear to have little functional value during rapid compensatory movements. Lateral destabilization complicates the control of compensatory stepping, a finding that may be particularly relevant to the problem of falls and hip fractures in elderly people. Older adults appear to have problems in controlling lateral stability when stepping to recover balance, even when responding to anteroposterior perturbation. Increased understanding and awareness of change-in-support reactions should lead to development of new diagnostic and therapeutic approaches for detecting and treating specific causes of imbalance and falling in elderly people and in patients with balance impairments.

Adaptation, Physiological↗

MDR 1 activation is the predominant resistance mechanism selected by vinblastine in MES-SA cells.

Single-step selection with vinblastine was performed in populations of the human sarcoma cell line MES-SA, to assess cellular mechanisms of resistance to the drug and mutation rates via fluctuation analysis. At a stringent selection with 20 nM vinblastine, resulting in 5-6 logs of cell killing, the mutation rate was 7 x 10(-7)per cell generation. Analysis of variance supported the hypothesis of spontaneous mutations conferring vinblastine resistance, rather than induction of adaptive response elements. Surviving clones displayed a stable multidrug resistance phenotype over a 3-month period. All propagated clones demonstrated high levels of resistance to vinblastine and paclitaxel, and lower cross-resistance to doxorubicin and etoposide. Activation of MDR 1 gene expression and P-glycoprotein function was demonstrable in all clones. No elevation was found in the expression of the mrp gene, the LRP-56 major vault protein and beta-tubulin isotypes (M40, beta4, 5beta, and beta9) in these mutants. We conclude that initial-step resistant mechanism in these vinblastine-selected mutants commonly arises from a stochastic mutation event with activation of the MDR 1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Reciprocal mitotic recombination is the predominant mechanism for the loss of a heterozygous gene in Saccharomyces cerevisiae.

The loss of a functional copy of a heterozygous tumor suppressor gene represents an important step during neoplastic transformation. In order to learn more about the genetic events that lead to spontaneous and drug-induced loss of heterozygosity, a diploid Saccharomyces cerevisiae strain was constructed that allows the detection of the loss of a heterozygous gene by means of direct selection. The strain contains a single functional URA3 gene copy inserted at the ADE2 locus located on the right arm of chromosome 15. In addition, the chromosome contains two other phenotypic marker genes, HIS3 which is located distal from URA3, and PHO80 which is closely linked to the centromere. The homologous chromosome lacks all three marker genes. Loss of the heterozygous copy of URA3 can easily be detected by 5-fluoro-orotic acid resistance of the resulting clones. Simple phenotypic tests of the resistant clones further allows one to distinguish whether the loss of the URA3 gene copy occurred by crossing over, chromosomal loss, or point mutation and gene conversion. Loss of heterozygosity was found to be induced in a dose-dependent fashion by UV radiation and by several chemical agents. All the tested mutagens induced loss of heterozygosity predominantly by crossing over.

Acid Phosphatase↗