Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.

Outcome of the acute abdomen in patients with previous spinal cord injury

Background: Patients with spinal cord injury (SCI) have always posed difficulties for the diagnosis of an acute abdomen. The aim of the present study was to define this problem retrospectively at Princess Alexandra Hospital and to assess the results of treatment for these patients. Methods: A retrospective review was conducted of 133 SCI patients admitted with an acute abdomen in the 16 years prior to this analysis at the Spinal Injuries Unit (SIU) of Princess Alexandra Hospital. There were 21 patients who conformed to the study criteria. All the patients had sustained traumatic SCI at or above the level of T11, more than 1 month prior to admission. Results: There were 13 male and eight female patients. The time lapse between SCI and the onset of an acute abdomen ranged from 1.5 months to 27 years. The age range was 26-79 years. The majority of patients had C6 injuries (six patients). There were 18 patients with injury levels above T6 and three patients with injuries below this level. The time taken to diagnose the cause of the acute abdomen ranged between 1 day and 3 months. Investigations were found to be useful in making the diagnoses in 61.9% of cases. There were 14 patients who had surgical interventions. Five patients had surgical complications and there were two deaths in the study. The length of follow up was 1-132 months. The mortality in the study was 9.5%. Conclusion: An aggressive approach to the diagnosis and treatment of the acute abdomen in SCI patients with suspicious symptoms is recommended. A high index of suspicion should be maintained in those patients with pre-existing SCI who present with abdominal trauma.

Phyllodes tumors of the breast: Correlation of nucleolar organizer regions with histopathological malignancy grading, flow cytometric DNA analysis and clinical outcome

To examine whether nucleolar organizer regions detected by argyrophilia (Ag-NOR counts) can be used as a prognostic indicator in phyllodes tumors of the breast, and to compare its usefulness with that of DNA flow cytometric analysis, 28 cases of breast phyllodes tumors (including 15 benign, two borderline and 11 malignant tumors) were subjected to Ag-NOR staining and counting as well as DNA flow cytometric analysis. S-phase fraction and DNA ploidy analysis showed useful trends for improving outcome predictions in malignant phyllodes tumors. However, high Ag-NOR counts were significant in predicting survival status (P = 0.013) and reached near statistical significance in predicting survival times (P = 0.07). In predicting survival status, results for Ag-NOR counts were significantly better than those for ploidy analysis (P = 0.02) and S-phase fraction (P < 0.01). Only S-phase fraction was significantly predictive of survival times (P = 0.025). It is concluded that Ag-NOR counts and DNA flow cytometric analysis, easily performed using paraffin sections, give information that can improve predictions made by histopathological classification. Ag-NOR counts are significant in predicting survival in the presence of histopathological features of malignancy.

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine

The beta -amino acid, taurine, is a full agonist of the human glycine receptor al subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta -amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta -amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta -amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alpha beta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alpha beta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alpha beta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alpha beta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin las an allosterically acting 'competitive' antagonist) binds to this residue.

The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase

Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane microlocalization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC 12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

Treatment outcome in individuals with chronic pain: is the Pain Stages of Change Questionnaire (PSOCQ) a useful tool? Jenny StrongCorresponding Author Contact Information, E-mail The Corresponding Author, a, Kym Westburya, Glen Smithb, Ian McKenziec and William Ryan

The efficacy of psychological treatments emphasising a self-management approach to chronic pain has been demonstrated by substantial empirical research. Nevertheless, high drop-out and relapse rates and low or unsuccessful engagement in self-management pain rehabilitation programs have prompted the suggestion that people vary in their readiness to adopt a self-management approach to their pain. The Pain Stages of Change Questionnaire (PSOCQ) was developed to assess a patient's readiness to adopt a self-management approach to their chronic pain. Preliminary evidence has supported the PSOCQ's psychometric properties. The current study was designed to further examine the psychometric properties of the PSOCQ, including its reliability, factorial structure and predictive validity. A total of 107 patients with an average age of 36.2 years (SD = 10.63) attending a multi-disciplinary pain management program completed the PSOCQ, the Pain Self-Efficacy Questionnaire (PSEQ) and the West Haven-Yale Multidimensional Pain Inventory (WHYMPI) pre-admission and at discharge from the program. Initial data analysis found inadequate internal consistencies of the precontemplation and action scales of the PSOCQ and a high correlation (r = 0.66, P < 0.01) between the action and maintenance scales. Principal component analysis supported a two-factor structure: 'Contemplation' and 'Engagement'. Subsequent analyses revealed that the PSEQ was a better predictor of treatment outcome than the PSOCQ scales. Discussion centres upon the utility of the PSOCQ in a clinical pain setting in light of the above findings, and a need for further research. (C) 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Debiasing the outcome effect: The role of instructions in an audit litigation setting

The outcome effect occurs where an evaluator, who has knowledge of the outcome of a judge's decision , assesses the quality of the judgment of that decision maker. If the evaluator has knowledge of a negative outcome, then that knowledge negatively influences his or her assessment of the ex ante judgment. For instance, jurors in a lawsuit brought against an auditor for alleged negligence are informed of an undetected fraud, even though an unqualified opinion was issued. This paper reports the results of an experiment in an applied audit judgment setting that examined methods of mitigating the outcome effect by means of instructions. The results showed that simply instructing or warning the evaluator about the potential biasing effects of outcome information was only weakly effective. However, instructions that stressed either (1) the cognitive non-normativeness of the outcome effect or (2) the seriousness and gravity of the evaluation ameliorated the effect significantly. From a theoretical perspective, the results suggest that there may both motivational and cognitive components to the outcome effect. In all, the findings suggest awareness of the outcome effect and use of relatively nonintrusive instructions to evaluators may effectively counteract the potential for the outcome bias.