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PCNA: structure, functions and interactions.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This toroidal-shaped protein encircles DNA and can slide bidirectionally along the duplex. One of the well-established functions for PCNA is its role as the processivity factor for DNA polymerase delta and epsilon. PCNA tethers the polymerase catalytic unit to the DNA template for rapid and processive DNA synthesis. In the last several years it has become apparent that PCNA interacts with proteins involved in cell-cycle progression which are not a part of the DNA polymerase apparatus. Some of these interactions have a direct effect on DNA synthesis while the roles of several other interactions are not fully understood. This review summarizes the structural features of PCNA and describes the diverse functions played by the protein in DNA replication and repair as well as its possible role in chromatin assembly and gene transcription. The PCNA interactions with different cellular proteins and the importance of these interactions are also discussed.

Animals↗

Factors responsible for the establishment of the body plan in the amphibian embryo.

A central topic of embryology is the establishment of the body plan during embryogenesis. Starting with maternal factors distributed in the early cleavage stages in distinct patterns and gradients cell-to-cell interactions including early embryonic induction result in the formation of mesoderm and the organizer area. While many facts are known about the role of growth factors like activin (closely related to the vegetalizing factor), processed Vg1, BMPs and FGF for mesoderm formation, the establishment of the central nervous system is not yet well understood. However, there is growing evidence that neural induction is a multistep process at the level of the dorsal mesoderm (organizer) and the reacting neuroectoderm. Therefore the existence of only one neuralizing factor is unlikely. We report about data that follistatin protein is not a direct neural inducer. Furthermore our comparative studies of Xenopus and Triturus exogastrulae indicate that planar signals are unlikely in the Triturus embryo (urodeles) during the early steps of neural induction. Vertical signals emanating from the chordamesoderm are essential for the terminal neuralization and regionalization of the central nervous system during gastrulation for both Xenopus and Triturus. The putative role of neuralizing factors and BMP/activin-like molecules for the stabilization or shift of neuroectoderm into different pathways of differentiation (epidermis or neural default state) is discussed.

Amphibians↗

Cognitive constraints on ordering operations: the case of geometric analogies.

Many tasks (e.g., solving algebraic equations and running errands) require the execution of several component processes in an unconstrained order. The research reported here uses the geometric analogy task as a paradigm case for studying the ordering of component processes in this type of task. In solving geometric analogies by applying mental transformations such as rotate, change size, and add a part, the order of performing the transformations is unconstrained and does not in principle affect solution accuracy. Nevertheless, solvers may bring cognitive constraints with them to the analogy task that influence the ordering of the transformations. First, we demonstrate that solvers have a preferred order for performing mental transformations during analogy solution. We then investigate three classes of explanations for the preferred order, one based on general information processing considerations, another based on task-specific considerations, and a third based on individual differences in analogy ability. In the first and third experiments, college students solved geometric analogies requiring two or three transformations and indicated the order in which they performed the transformations. There was close agreement on nearly the same order for both types of analogies. In the second experiment, subjects were directed to perform pairs of transformations in the preferred or unpreferred order. Both speed and accuracy were greater for the preferred orders, thus validating subjects' reported orders. Ability differences were observed for only the more difficult three-transformation problems: High- and middle-ability subjects agreed on an overall performance order, but the highs were more consistent in their use of this order. Low-ability subjects did not consistently order the transformations for these difficult problems. The general information processing factor examined was working-memory load. A number of task factors have been shown to affect working-memory load during the solution of inductive reasoning problems. Of these, we chose to examine process difficulty. Because analogies are solved in working memory, performing more difficult transformations earlier may reduce working-memory load and facilitate problem solution. However, the observed performance order was not correlated with transformation difficulty. The first task-specific factor considered was that some transformations may be identified earlier, possibly because of perceptual salience, and that the performance order follows the identification order.(ABSTRACT TRUNCATED AT 400 WORDS)

Cognition↗

Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity.

Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells. It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates. This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC () binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA. In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors. Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA. Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit subcomplex. The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex. In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.

Adenosine Triphosphatases↗

Factors affecting medication-order processing time.

The factors affecting medication-order processing time at one hospital were studied. The order processing time was determined by directly observing the time to process randomly selected new drug orders on all three work shifts during two one-week periods. An order could list more than one drug for an individual patient. The observer recorded the nature, location, and cost of the drugs ordered, as well as the time to process the order. The time and type of interruptions also were noted. The time to process a drug order was classified as six dependent variables: (1) total time, (2) work time, (3) check time, (4) waiting time I--time from arrival on the dumbwaiter until work was initiated, (5) waiting time II--time between completion of the work and initiation of checking, and (6) waiting time III--time after the check was completed until the order left on the dumbwaiter. The significant predictors of each of the six dependent variables were determined using stepwise multiple regression. The total time to process a prescription order was 58.33 +/- 48.72 minutes; the urgency status of the order was the only significant determinant of total time. Urgency status also significantly predicted the three waiting-time variables. Interruptions and the number of drugs on the order were significant determinants of work time and check time. Each telephone interruption increased the work time by 1.72 minutes. While the results of this study cannot be generalized to other institutions, pharmacy managers can use the method of determining factors that affect medication-order processing time to identify problem areas in their institutions.

Medication Systems, Hospital↗

Return to work experience of injured workers in a case management program.

A major goal of case management programs is the worker's timely return to work. Few studies have examined return to work from the perspective of the injured worker. This article describes the findings from the case management evaluation that describe the return to work experience of workers who sustained catastrophic injuries, or who had secondary conditions or complications following the injury occurrence. Among the factors determined to affect the return to work experience were structural factors (i.e., psychosocial variables including job satisfaction and relationship with employer and coworkers, financial pressures, and system issues such as securing benefits) and process factors (i.e., interaction with service providers and with the workers' compensation system). Outcomes are described in terms of satisfaction with services and return to work.

Absenteeism↗

Treatment process and relapse to opioid use during methadone maintenance.

A general framework for studying drug abuse treatment process factors is presented, and components are then used to predict relapse to opioid use during treatment in methadone maintenance. Major domains of the treatment process research framework include client variables at entry, program characteristics, treatment events, and client outcomes. The analyses rely on the use of proportional hazards models to identify significant outcome predictors in a sample of 590 methadone maintenance clients from 21 clinics in the Research Triangle Institute/Treatment Outcome Prospective Study (RTI/TOPS) data system who remained in treatment at least 3 months. The analyses were performed on the total sample and separately on clients from three groups of clinics classified on the basis of the distribution of client relapse rates and tenure in treatment. Relapse rates were related to dosage level, client monitoring with urinalyses, and methadone take-home privileges in some clinics, and hence, these time-varying treatment events were important factors in treatment outcomes. Even at entry to treatment, some measures were found to be related to how the client later performed during treatment. Finally, it was also found that the particular area of professional speciality of the staff making client diagnosis at intake and preparing treatment plans was associated with client outcomes.

Adult↗

Explanatory model for the interaction of factors in the caries process.

The interaction of factors in the caries process has recently been discussed in a number of articles. Based on this, a new explanatory model has been constructed, consisting of an equilateral triangle with one of the apices on a horizontal line, along which dietary and oral hygiene habits are scaled. The triangle represents the total area of interaction. A horizontal line of resistance cuts off a lower part corresponding to the area within which caries does not occur. Through planogeometric calculations the remaining part of the triangle can be divided into areas, approximately corresponding to the relative caries values of various combinations of dietary and oral hygiene habits. The model has proved to be a useful instrument for demonstrating and creating an understanding of interactive processes among various groups of dental health personnel. The example presented is based on a pilot study of 55 4-year-old children in Malmö.

Child, Preschool↗

Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

Amino Acid Sequence↗

SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements.

Interphase chromatin is arranged into topologically separated domains comprising gene expression and replication units through genomic sequence elements, so-called MAR or SAR regions (for matrix- or scaffold-associating regions). S/MAR regions are located near the boundaries of actively transcribed genes and were shown to influence their activity. We show that scaffold attachment factor B (SAF-B), which specifically binds to S/MAR regions, interacts with RNA polymerase II (RNA pol II) and a subset of serine-/arginine-rich RNA processing factors (SR proteins). SAF-B localized to the nucleus in a speckled pattern that coincided with the distribution of the SR protein SC35. Furthermore, we show that overexpressed SAF-B induced an increase of the 10S splice product using an E1A reporter gene and repressed the activity of an S/MAR flanked CAT reporter gene construct in vivo . This indicates an association of SAF-B with SR proteins and components of the transcription machinery. Our results describe the coupling of a chromatin organizing S/MAR element with transcription and pre-mRNA processing components and we propose that SAF-B serves as a molecular base to assemble a 'transcriptosome complex' in the vicinity of actively transcribed genes.

3T3 Cells↗

Participation of the nuclear cap binding complex in pre-mRNA 3' processing.

Communication between the 5' and 3' ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5' end is bound by the nuclear cap binding complex (CBC). This RNA-protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3' end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5' and 3' ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3' processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of "closed-loops," due to interactions between factors bound at their 5' and 3' ends.

Cell Nucleus↗

Influence of the primary anticoagulant on the recovery of factor VIII in cryoprecipitate.

The influence of anticoagulant on overall factor VIII-yield was measured by drawing blood from one donor simultaneously in three bags containing ACD, CPD and heparin, respectively. After parallel processing factor VIII:C and factor VIII:CAg were measured. It is concluded that, under the circumstances used in this experiment, CPD gives the highest yield of factor VIII.

Adenine↗

Processing of eukaryotic pre-rRNA: the role of the transcribed spacers.

The 17-18S, 5.8S, and 25-28S rRNA species of eukaryotic cells are produced by a series of nucleolytic reactions that liberate the mature rRNAs from the large primary precursor transcript synthesized by RNA polymerase 1. Whereas the order of the cleavage reactions has long been established, until recently little information was available on their molecular details, such as the nature of the proteins, including the nucleolytic enzymes, involved and the signals directing the processing machinery to the correct sites. This situation is now rapidly changing, in particular where yeast is concerned. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has considerably enhanced our knowledge of cis-acting structural features within the pre-rRNA, in particular the transcribed spacer sequences, that are critical for correct and efficient removal of these spacers. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this paper, we will focus predominantly on the nature and role of the cis-acting processing elements as identified in the transcribed spacer regions of Saccharomyces cerevisiae pre-rRNA.

Base Sequence↗

Case management services for injured workers. Providers' perspectives.

The perceptions of service providers involved in case management services are described here. The service providers included claims managers, occupational nurse consultants, attending physicians, and nurse case managers. The purposes of this phase of the study were to describe these providers' perceptions about the case management program; to organize findings according to the quality assessment model that guided this study (including structure, function, and outcomes); and to identify barriers and facilitators to satisfaction with case management services. Structural factors that affected services included the workers' compensation system, construction of the service team, roles within the program, and individual attributes of service providers. Process factors were conflicts among parties, role of communication, and interaction with workers. Outcomes were described in terms of program efficiency and effectiveness. Data are used to illustrate and explain each of these themes.

Accidents, Occupational↗

[The principles of the combined and simultaneous action of industrial factors].

The laws of the development of tension of the main systems and regulatory mechanisms of the organism of people working both under exposure to occupational factors altogether and to various combinations of ergometric indices of work. Joint action of unfavourable labour conditions (dust, industrial noise, insufficient lighting) and of working process factors (local muscular stress, hardness and tension of labour), as a rule, cause potentiation of unfavourable changes in the functional status of the organism. In the majority of occupational groups the factors studied predetermine in 76-96% of cases changes in the functional state of the major systems of the workers' organism.

Humans↗

Application of monoliths for downstream processing of clotting factor IX.

In this paper, the application of monolithic columns for downstream processing of different clotting factor IX concentrates is shown. Determination of basic chromatographic conditions as well as investigations on the regeneration of disk- and tube-shaped monolithic columns using human serum albumin as a model protein, were performed. Separation of factor IX and vitronectin, a possible impurity in commercial factor IX concentrates was accomplished using disk-shaped monolithic columns. These same applications were also carried out with identical results on up-scaled tube-shaped monolithic columns. Since these media allow very fast separations, this method can be successfully applied not only to an in-process control of the purification of factor IX but also to other biopolymers from human plasma. Besides, the same application on the up-scaled tube-shaped monolithic column was successfully carried out.

Biopolymers↗

Quality assurance for psychiatric emergencies. An analysis of assessment and feedback methodologies.

Improvement in the quality of evaluation and treatment of behavioral emergencies presenting to hospital emergency rooms requires a multicomponent assessment of resources, clinician performance, and outcome. The authors present a summary of data and conclusions derived from index-based assessment of quality of care in over 2000 psychiatric emergencies. The study revealed that emergency room physicians rendered high-quality medical care, but were deficient in crucial aspects of psychiatric evaluation and treatment including evaluation of dangerousness and substance abuse and provision of appropriate follow-up care. A positive association was observed between quality of care and compliance with referral. Subsequent review with the staff pointed to issues such as staffing, emergency room and community resources, and adequacy of documentation as significant influences on quality of care, as assessed by the chart-audit, index approach. A pilot study demonstrated uniform improvement of quality scores in a small group of physicians receiving written and oral feedback about their performance. The use of focused quality assessment studies that examine both structural and process factors, and their correlation with outcome, is discussed in light of the information obtained from the previously described study. A feedback methodology that emphasizes an interactive process to identify deficiencies in care and to explore their causes and remedies is presented.

Aftercare↗

A C-terminal domain, which prevents secretion of the neuroendocrine protein 7B2 in Saccharomyces cerevisiae, inhibits Kex2 yet is processed by the Yap3 protease.

Recent reports reveal that the C-terminal half of the neuroendocrine polypeptide 7B2 selectively inhibits and binds PC2, a mammalian prohormone converting enzyme that is homologous to the yeast pro-alpha-factor processing protease Kex2. During attempted secretion of the 185 amino-acid human 7B2 in Saccharomyces cerevisiae, we observe that the protein is mostly retained inside the cell. However a mutant polypeptide (7B2 delta 1), where the C-terminal 48 amino acids of 7B2 are deleted, is efficiently secreted. Two shorter C-terminal truncations either permit poor secretion or no secretion at all. Surprisingly, full-length 7B2 but not 7B2 delta 1 abolishes the catalytic activity of Kex2, indicating that C-terminal residues of 7B2 might also be important for inhibition of the yeast protease. When the KEX2 gene is disrupted, yeast cells unexpectedly secrete a 7B2 variant similar in size to 7B2 delta 1, suggesting involvement of the alternate yeast prohormone convertase Yap3 in processing. Secretion is enhanced by overexpression of Yap3 and by the presence of a Lys-Arg residue at the processing site of precursor 7B2. These results purport that, in neuroendocrine cells too, secretion of 7B2 could be mediated by a homologue of Yap3.

Aspartic Acid Endopeptidases↗