Stabilization of native protein fold by intein-mediated covalent cyclization

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10degreesC by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG = 2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding I measured by stopped-flow circular dichroism. rate of less than 4 min(-1) The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E. coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding. (C) 2005 Elsevier Ltd. All rights reserved.

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation Of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean N-15-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

A new social parasite in the ant genus Ectatomma F. Smith (Hymenoptera, Formicidae, Ectatomminae)

Ectatomma parasiticum Feitosa & Fresneau, a new ant species socially parasitic on Ectatomma tuberculatum (Olivier), is described from gynes discovered in Apazapan, state of Veracruz, Mexico, and reared in the laboratory. Ectatomma parasiticum is the first social parasite described in the Ectatomminae. This species can be distinguished from its host by morphological and behavioral features characteristic of the inquilines known in other ant subfamilies including reduced size, thickened petiole, and agonistic interactions with host species.

Injury and alcohol: a hospital emergency department study

A pilot survey was undertaken of injury presentations to a public hospital emergency department to determine patterns of alcohol use in this population. Of the 402 injury presentations in the study period, a total of 236 injury cases were interviewed, of whom 45% (n=107) and 29% (n=69) had consumed alcohol 24 and 6 hours prior to injury. Mean age for all injury presentations was 35.1 years, and 32.6 years for alcohol injury cases. For both injury groups, males were significantly younger than females. Recent alcohol ingestion was three times more common among male than female injury presentations, but with females drinking at significantly lower levels. Of males who had consumed alcohol 6 hours prior to injury, nearly 70% were drinking at NHMRC harmful levels and 61% had drunk more than eight standard drinks. Overall, alcohol-involved injury cases commonly occurred among low-income, single males around 30 years of age who were regular heavy drinkers who were drinking heavily in licensed premises prior to their injury, and who sustained injury through intentional harm. In addition, one in five of the alcohol-involved injury cases were aged 15-18 years, i.e. below the legal age of purchase. The high proportion of hazardous and harmful drinkers among those who had consumed alcohol within the last 6 hours, and the injury sample overall, highlights the need for further research to explore the relationship between the occurrence of injury and the drinking patterns and environments associated with injury. Further research is also required to assess the efficacy of early and brief interventions for alcohol and drug use within the emergency ward setting. This information would enable appropriate public health interventions to be initiated.

Icodextrin as salvage therapy in peritoneal dialysis patients with refractory fluid overload

Background Icodextrin is a high molecular weight, starch-derived glucose polymer, which is capable of inducing sustained ultrafiltration over prolonged (12–16 hour) peritoneal dialysis (PD) dwells. The aim of this study was to evaluate the ability of icodextrin to alleviate refractory, symptomatic fluid overload and prolong technique survival in PD patients. Methods A prospective, open-label, pre-test/post-test study was conducted in 17 PD patients (8 females/9 males, mean age 56.8 ± 2.9 years) who were on the verge of being transferred to haemodialysis because of symptomatic fluid retention that was refractory to fluid restriction, loop diuretic therapy, hypertonic glucose exchanges and dwell time optimisation. One icodextrin exchange (2.5 L 7.5%, 12-hour dwell) was substituted for a long-dwell glucose exchange each day. Results Icodextrin significantly increased peritoneal ultrafiltration (885 ± 210 ml to 1454 ± 215 ml, p < 0.05) and reduced mean arterial pressure (106 ± 4 to 96 ± 4 mmHg, p < 0.05), but did not affect weight, plasma albumin concentration, haemoglobin levels or dialysate:plasma creatinine ratio. Diabetic patients (n = 12) also experienced improved glycaemic control (haemoglobin Alc decreased from 8.9 ± 0.7% to 7.9 ± 0.7%, p < 0.05). Overall PD technique survival was prolonged by a mean of 11.6 months (95% CI 6.0–17.3 months). On multivariate Cox proportional hazards analysis, extension of technique survival by icodextrin was only significantly predicted by baseline net daily peritoneal ultrafiltration (adjusted HR 2.52, 95% CI 1.13–5.62, p < 0.05). Conclusions Icodextrin significantly improved peritoneal ultrafiltration and extended technique survival in PD patients with symptomatic fluid overload, especially those who had substantially impaired peritoneal ultrafiltration.

Quality Assurance Through a Continuous Curriculum Review (CCR) Strategy: Reflections on a Pilot Project

Institutional research can be defined as "the activity in which the research effort of an academic institution is directed at the solution of its own problems and to the enhancement of its own performance" (Woodward, 1993, p. 113). This paper describes and reflects on an attempt at the University of Queensland to address the need for course quality appraisal for improvement. The strategy, Continuous Curriculum Review (CCR) is simply an attempt to trial and promote regular comprehensive data collection for developing 'snapshot' views of whole curricula so that decisions about what to change and what to change first can be made in an empirically defensible and timely manner. The strategy and reporting protocols that were developed are described, and the costs and benefits of engaging in this kind of data gathering exercise for quality assurance and quality enhancement purposes are discussed.

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.

Melanocytic nevi, solar keratoses, and divergent pathways to cutaneous melanoma

Background: Some melanomas form on sun-exposed body sites, whereas others do not. We previously proposed that melanomas at different body sites arise through different pathways that have different associations with melanocytic nevi and solar keratoses. We tested this hypothesis in a case-case comparative study of melanoma patients in Queensland, Australia. Methods: We randomly selected patients from among three prespecified groups reported to the population-based Queensland Cancer Registry: those with superficial spreading or nodular melanomas of the trunk (n = 154, the reference group), those with such melanomas of the head and neck (n = 77, the main comparison group), and those with lentigo maligna melanoma (LMM) (n = 75, the chronic sun-exposed group). Each participant completed a questionnaire, and a research nurse counted melanocytic nevi and solar keratoses. We calculated exposure odds ratios (ORs) and 95% confidence intervals (CIs) to quantify the association between factors of interest and each melanoma group. Results: Patients with head and neck melanomas, compared with patients with melanomas of the trunk, were statistically significantly less likely to have more than 60 nevi (OR = 0.34, 95% CI = 0.15 to 0.79) but were statistically significantly more likely to have more than 20 solar keratoses (OR = 3.61, 95% CI = 1.42 to 9.17) and also tended to have a past history of excised solar skin lesions (OR = 1.87, 95% CI = 0.89 to 3.92). Patients with LMM were also less likely than patients with truncal melanomas to have more than 60 nevi (OR = 0.32, 95% CI = 0.14 to 0.75) and tended toward more solar keratoses (OR = 2.14, 95% CI = 0.88 to 5.16). Conclusions: Prevalences of nevi and solar keratoses differ markedly between patients with head and neck melanomas or LMM and patients with melanomas of the trunk. Cutaneous melanomas may arise through two pathways, one associated with melanocyte proliferation and the other with chronic exposure to sunlight.

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to-exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.

Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLA181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP-conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps(-) phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.