Gagging and cooperation in 4-12-year-old children over a series of dental appointments.

PURPOSE: The purposes were to evaluate if gagging can affect children's cooperation with treatment, investigate possible changes in gagging and study factors that can predict children's behaviour over dental treatment. METHODS: 255 children aged 4-12 years, needing at least three consecutive dental appointments, completed the Children's Fear Survey Schedule-Dental Subscale before examination. The Gagging Problem Assessment was performed before the initial examination and after the third/final appointment. Frankl's Behaviour Rating Scale (FBRS) was used to rate children's behaviour. Multivariate Mixed Linear and Logistic Regression Models were used. RESULTS: Children with definitely positive behaviour were significantly older, less fearful and less likely to gag before treatment. Girls and older children had lower odds of gagging over time, while fearful children had higher odds. There were no statistically significant associations between gagging over time, FBRS and the type of dental treatment at the third appointment. The percentage of children who gagged after the third appointment (27.05%) was significantly lower as compared to the percentage found at the initial appointment (32.54%; p = 0.004). CONCLUSION: Among the variables studied, age, dental fear, initial GPA and type of treatment were good predictors of children's behaviour during dental treatment.

Factors associated with gagging during radiographic and intraoral photographic examinations in 4-12-year-old children.

AIMS: No studies are available in paediatric samples evaluating gagging during toothbrushing, radiographic and/or intraoral photographic examinations. The aims were to collectively examine potential factors associated with gagging during radiographs and intraoral photographs in 4-12-year-old children. METHODS: Parents/guardians of 395 children (aged 4-12 years old) completed questionnaires asking about their children's toothbrushing habits. Children completed Greek versions of the Gagging Assessment Scale (GAS) and the Children's Fear Survey Schedule-Dental Subscale (CFSS-DS), while the dentist used the shorter version of the Gagging Problem Assessment (GPA-de-c/SF) to objectively assess gagging. X-ray and Photo Rating Scales were created to evaluate gagging during X-rays and photographs, respectively. Multivariate logistic regression was used to examine the relationship between the potential factors and gagging. RESULTS: 59 of 275 patients (21%) and 56 of 276 patients (20%) who needed X-rays and intraoral photographs, respectively, gagged. Children who gagged during X-rays had significantly higher GAS scores (p = 0.007). Boys, younger children, and those who gagged on GPA-de-c/SF were more likely to gag during X-rays, and children who gagged on GPA-de-c/SF were more likely to gag during photographs. Brushing habits were not related to dental fear or gagging. CONCLUSION: Of the variables which we studied, GPA-de-c/SF most strongly affected the odds of gagging during taking radiographs and/or intraoral photographs.

Assessing anxiety symptoms in children: Agreement between parents and children.

Assessment of anxiety disorders in children is a difficult process and requires the use of multiple resources of information. Such resources may come from children, parents, and educators and they also require the use of multiple types of diagnostic tools, like structured and semi-structured clinical interviews, as well as self-report questionnaires. Previous research shows that anxiety symptoms ratings of different informants are to some degree correlated (low to moderate agreement) but nonetheless also often show clear discrepancies. Important variables may affect the degree of child-parent agreement. The present study focused on child's gender and age possible impact. The aim of the present study was to examine the agreement between children's and parents' reports on self-reported questionnaires for anxiety symptoms. 431 children from 4th to 6th grade of elementary school and their parents participated in the study. 190 were boys and 241 were girls. Both children and their parents responded to Spence Children's Anxiety Scale (SCAS) (child's and parent's version correspondingly). Relations between children's and parents' reports concerning anxiety symptoms were examined by calculation Pearson's correlation coefficients. The results showed that there was a medium but statistically important positive correlation between children's and parents' reports on SCAS total score (r=0.50, p<0.01). Concerning SCAS subscales results supported that higher correlations were those reported for Separation Anxiety (r=0.53, p<0.01) and Fear of Physical Injury (r=0.55, p<0.01). Concerning gender differences the present study found that correlation coefficients for girls were higher than for boys in SCAS total score (r=0.57 and r=0.39 correspondingly, p<0.01). Correlations according to age showed that the highest correlation coefficients were found in comparatively older children (r=0.34, r=0.54 και r=0.63, p<0.01 for 4th, 5th and 6th Grade). The latter underlies that in the process of assessing and diagnosing anxiety disorders in children, it is both necessary and important to gather information from multiple sources, especially in cases of younger children.

Low oxygen tension enhances hepatitis C virus replication.

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

[A study of the effectiveness of a group-based cognitive-behavioral parent training program].

The role of the family in the development of the child as well as the quality of the parent-child relationship and its effect in the social, mental and cognitive development of the child has been the focus of attention of many sciences and scientists and it has been discovered that many parents are not well prepared to do their best for their children. The parent training programmes are willing to partly give a solution to this with their preventive role. In recent years, the effectiveness of the parent training programmes, which are offered to "high risk" parents, has been the focus of a big amount of research, meta-analyses and reviews. A smaller amount concerns the effectiveness of the universal programmes which are offered to the parents of the general population. The effectiveness of a ten-meeting structured group parent training programme of cognitive-behavioral approach, which had been offered to mothers of the general population, was researched in the present study. It aimed to research the effectiveness of the specific programme in the children's behavior and the subjective perception of the functionality of the family of the mothers who chose to participate in and completed the programme (n=56, experimental group/participants), compared to those who chose not to (n=113, control group/non participants). The mothers of the two groups were mothers with children aged between 2 and 12 and filled in the Family Adaptation and Cohesion Scales, FACES-III and the Questionnaire of Inter-personal and Cross-personal Adaptation, before (Phases A) and after (Phases B) the programme. The two groups were fully matched and did not present any significant difference regarding their demographic characteristics. During both Phases A and B of the training programme participants and non-participants expressed a high degree of satisfaction by the functionality of their family and did not differentiate significantly in the evaluation of the existent family cohesion and adaptability, the type of the family based on the cohesion and adaptability and the general type of family based on the functionality. In addition, while the children of the participants were, before the start of the programme, in a significantly disadvantaged position compared to the children of the non-participants, after the end of the programme, they were significantly improved, decreasing the negative symptoms and behaviors. This particular parent training programme of cognitive-behavioral approach, as well as other programmes which belong to the same theoretical direction, could contribute to the prevention of the behavior problems and the promotion of the mental health.

Hypoxia inducible factor-1 alpha and vascular endothelial growth factor in biopsies of small cell lung carcinoma.

Neoangiogenesis has been documented in small cell lung carcinoma (SCLC). In addition, antiangiogenic therapies are being tested in clinical trials that involve SCLC. However, study of the underlying mechanisms has been performed almost exclusively in cell lines. In the current study, we immunostained 30 biopsy samples of SCLC with antibodies to hypoxia inducible factor-1 alpha (HIF-1 alpha), vascular endothelial growth factor (VEGF), vascular endothelial growth factor-receptor 1 (VEGF-R1/flt-1) and vascular endothelial growth factor-receptor 2 (VEGF-R1/flk-1). The immunoreactivity was analyzed using a bivariate Spearman correlation test and linear regression analysis. We found significant correlation between HIF-1 alpha nuclear staining and VEGF staining. Moreover HIF-1 alpha+/VEGF+ cases were associated with poor survival. We also found a positive correlation between VEGF and VEGF-R2 expression. We suggest that a HIF-1 alpha/VEGF angiogenic pathway may exist in vivo in SCLC, similar to that in non-SCLC. Our data also suggest a potential VEGF/VEGFR-2 autocrine pathway in SCLC. The inclusion of novel inhibitors to HIF-1 alpha and other factors may optimize antiangiogenic interventions in SCLC.

Enzyme-linked immunosorbent assay for pharmacological studies targeting hypoxia-inducible factor 1alpha.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of a wide range of genes related to oxygen delivery and metabolic adaptation under hypoxic (low-oxygen) conditions. HIF-1 is, in fact, a heterodimer of two subunits, HIF-1alpha and HIF-1beta. The only analytical methods available for measuring HIF-1alpha levels in tumors are immunohistochemistry and Western blotting. Immunohistochemistry has the advantage of allowing the identification and direct examination of HIF-1alpha-expressing cells, but has the intrinsic limitation, as for Western blotting, of being nonquantitative. We developed and validated an enzyme-linked immunosorbent assay (ELISA) approach to measure HIF-1alpha levels in cultured tumor cell lines in vitro. HIF-1alpha was expressed in thirteen tumor cell lines grown under hypoxic conditions; however, the levels differed strongly between cell lines. These data point to intrinsic differences between cell lines for the induction of HIF-1alpha under hypoxic conditions. The ELISA developed in the present study is thus an interesting alternative to other analytical methods used to measure HIF-1alpha protein levels and should be useful in preclinical pharmacological studies targeting HIF-1alpha.

The intracellular location of two aminoacyl-tRNA synthetases depends on complex formation with Arc1p.

In yeast, two aminoacyl-tRNA synthetases, MetRS and GluRS, are associated with Arc1p. We have studied the mechanism of this complex formation and found that the non-catalytic N-terminally appended domains of MetRS and GluRS are necessary and sufficient for binding to Arc1p. Similarly, it is the N-terminal domain of Arc1p that contains distinct but overlapping binding sites for MetRS and GluRS. Localization of Arc1p, MetRS and GluRS in living cells using green fluorescent protein showed that these three proteins are cytoplasmic and largely excluded from the nucleus. However, when their assembly into a complex is inhibited, significant amounts of MetRS, GluRS and Arc1p can enter the nucleus. We suggest that the organization of aminoacyl-tRNA synthetases into a multimeric complex not only affects catalysis, but is also a means of segregating the tRNA- aminoacylation machinery mainly to the cytoplasmic compartment.

Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast.

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.

Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export.

The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP "core proteins" Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3' end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way.

Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs.

Eukaryotic aminoacyl-tRNA synthetases, in contrast to their prokaryotic counterparts, are often part of high molecular weight complexes. In yeast, two enzymes, the methionyl- and glutamyl-tRNA synthetases associate in vivo with the tRNA-binding protein Arc1p. To study the assembly and function of this complex, we have reconstituted it in vitro from individually purified recombinant proteins. Our results show that Arc1p can readily bind to either or both of the two enzymes, mediating the formation of the respective binary or ternary complexes. Under competition conditions, Arc1p alone exhibits broad specificity and interacts with a defined set of tRNA species. Nevertheless, the in vitro reconstituted Arc1p-containing enzyme complexes can bind only to their cognate tRNAs and tighter than the corresponding monomeric enzymes. These results demonstrate that the organization of aminoacyl-tRNA synthetases with general tRNA-binding proteins into multimeric complexes can stimulate their catalytic efficiency and, therefore, offer a significant advantage to the eukaryotic cell.

Assessment of dysfunctional working models of self and others in schizophrenic patients: a summary of data collected in nine nations. International Research Group.

OBJECTIVE: To investigate the cross-cultural feasibility of a new scale for assessing dysfunctional working models of self and others, and to evaluate its discriminative power. METHOD: Schizophrenic patients (N=351), non-psychotic patients (N= 86) and non-clinical subjects (N= 511) collected in 10 centres completed the DWM-S. Current psychopathology was assessed by means of the BPRS. RESULTS: Alpha coefficients were high in all samples. Mean scores on the DWM-S appeared to be comparable in all countries, suggesting cross-national generalizability. No significant correlation was found with sex, age, levels of psychopathology and duration of illness. Discriminant analyses showed that more than 70% of the schizophrenic patients are correctly classified. CONCLUSION: The DWM-S is an easily administered self-report instrument which allows to pinpoint internal dysfunctional working models of self and others in various types of patients. It is a useful tool for case conceptualization, especially when psychotherapeutic interventions are part of the treatment programme.

Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p.

Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S. cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.

Review: transport of tRNA out of the nucleus-direct channeling to the ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin beta superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.

An aminoacylation-dependent nuclear tRNA export pathway in yeast.

Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries.

Transfer RNA biogenesis: A visa to leave the nucleus.

Only correctly folded and mature tRNAs can leave the nucleus and enter the cytoplasm. Surprisingly, tRNA-aminoacylation has been found to occur, not only in the cytosol, but also inside the nucleus, where it may act as an additional proofreading step and facilitate the export of 'ready-to-function' aminoacyl-tRNAs.

A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants.

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA.

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.

A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases.

Two yeast enzymes that catalyze aminoacylation of tRNAs, MetRS and GluRS, form a complex with the protein Arc1p. We show here that association of Arc1p with MetRS and GluRS is required in vivo for effective recruitment of the corresponding cognate tRNAs within this complex. Arc1p is linked to MetRS and GluRS through its amino-terminal domain, while its middle and carboxy-terminal parts comprise a novel tRNA-binding domain. This results in high affinity binding of cognate tRNAs and increased aminoacylation efficiency. These findings suggest that Arc1p operates as a mobile, trans-acting tRNA-binding synthetase domain and provide new insight into the role of eukaryotic multimeric synthetase complexes.