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PubMed2004

Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity.

Carboxypeptidase M (CPM), an extracellular glycosylphosphatidyl-inositol(GPI)-anchored membrane glycoprotein belonging to the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, specifically removes C-terminal basic residues from peptides and proteins. Due to its wide distribution in human tissues, CPM is believed to play important roles in the control of peptide hormone and growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. We have crystallized human GPI-free CPM, and have determined and refined its 3.0A crystal structure. The structure analysis reveals that CPM consists of a 295 residue N-terminal catalytic domain similar to that of duck CPD-2 (but only distantly related to CPA/B), an adjacent 86 residue beta-sandwich C-terminal domain characteristic of the CPN/E family but more conically shaped than the equivalent domain in CPD-2, and a unique, partially disordered 25 residue C-terminal extension to which the GPI membrane-anchor is post-translationally attached. Through this GPI anchor, and presumably via some positively charged side-chains of the C-terminal domain, the CPM molecule may interact with the membrane in such a way that its active centre will face alongside, i.e. well suited to interact with other membrane-bound protein substrates or small peptides. Modelling of the C-terminal part of the natural substrate Arg(6)-Met-enkephalin into the active site shows that the S1' pocket of CPM is particularly well designed to accommodate P1'-Arg residues, in agreement with the preference of CPM for cleaving C-terminal Arg.

Amino Acid Sequence↗
PubMed2005

Persistence of inflammatory cytokines cause a spectrum of chronic progressive diseases: implications for therapy.

Chronic progressive disease (CPD) are the Western world's major cause of mortality (National Center for Health Statistics. Vol. 52. National vital 176 statistics reports. Deaths: final data for 2001; 2003, p. 3). Most chronic diseases present with symptoms and signs specific for the dysfunctional organ. Nonetheless, substantial commonalities are identifiable at the tissue and cellular level. These include a striking increase in inflammatory cytokines in both the tissue and the serum, accompanied by tissue destruction, apoptosis and tissue fibrosis. Individual inflammatory cytokines possess the capacity to induce these tissue effects in vitro and in vivo. Further, an elevation in systemic levels of cytokines and CRP predict an increase risk at all stages of these diseases. Therapies that inhibit the stimuli, the mediators or the responses to persistent inflammation appear to have an inhibitory effect on progression of specific diseases. We hypothesize that a unifying paradigm for CPD can be constructed based on these observations. Destabilizing stimuli activate the normal protective homeostatic inflammatory response in tissue. As the stimulus is eliminated and tissue heals, the inflammatory response recedes, re-establishing homeostasis. If inflammatory cytokines persist, however, both cell apoptosis and tissue fibrosis can be induced. Two aspects of evolution provide a potential mechanistic basis for this hypothesis. We may speculate that Darwinian selection favored early development of a system that channeled a broad spectrum of external and internal challenges through a generic response system. Thus, the mediators that respond to noxious stimuli became universal throughout organisms and species. Because these genetic responses were part of the DNA programming of all cells prior to differentiation, the tissue response to inflammation is also both uniform in nature, and narrow in scope. Subsequent cell differentiation resulted in vast differences in tissue function, so that organ dysfunction appears with the myriad symptoms and signs we recognize as individual diseases. Interference with inflammatory cytokines, e.g., with HMG Coa reductase inhibitors, inhibits a spectrum of chronic progressive diseases, independent of any LDL lowering effect.

Apoptosis↗
PubMed2006

Cell type and DNA damage specific response of human skin cells to environmental agents.

The epidermis has evolved to provide a barrier against the environment, which is essential for survival. This barrier is constituted and continuously regenerated by terminally differentiating keratinocytes. Here, we summarize the main features of the response to UVB and oxidizing agents of human keratinocytes and compare it with that of fibroblasts. Keratinocytes are more resistant to the lethal effects of UVB than fibroblasts and remove cyclobutane pyrimidine dimers (CPD) more efficiently than fibroblasts. UV photoproducts are repaired by the nucleotide excision repair (NER) system by two distinct sub-pathways: global genome repair (GGR) that repairs lesions on the genome overall, and transcription coupled repair (TCR) that operates on transcribed sequences of active genes. By using NER-defective cells we demonstrated that the improved repair of UVB damage by keratinocytes is due to a more efficient GGR. A defect in TCR was associated with a strong apoptotic response in fibroblasts but not in keratinocytes, whereas a defect in GGR had no effect on the apoptotic response of either cell type. We speculate that the persistence of CPD in the transcribed sequences triggers apoptosis in fibroblasts but not in keratinocytes where GGR operates as back-up system to remove transcription-blocking lesions. As observed for UVB, keratinocytes are also more resistant to the lethal effects of oxidizing agents than fibroblasts. We show that keratinocytes are characterized by a strong anti-oxidant capacity and a higher susceptibility to reactive oxygen species (ROS)-induced apoptosis than fibroblasts. All together these results provide a clear evidence that the response to environmental agents is strongly affected by the type of damage as well as by the cellular background.

Antioxidants↗
PubMed2005

Learning needs analysis: the development of a tool to support the on-going professional development of multiple sclerosis specialist nurses.

Continuous professional development (CPD) is essential in modern day nursing. Learning needs analysis (LNA) is an important element of CPD. This paper describes the development of a LNA tool for multiple sclerosis specialist nurses. The tool contains an empirically developed LNA schedule and uses a blend of both subjective and objective exercises to help the nurse formulate a comprehensive learning plan. The tool is organised into four phases: phase 1, focuses on the knowledge and skills necessary to the fulfillment of work-based objectives; phase 2, involves a more in-depth assessment of particular areas of professional knowledge; phase 3, examines learning strategies; and phase 4, involves the development of a learning plan. The tool has been implemented throughout the UK via local trainers. While the tool was developed for a specialist nurse population the principles upon which it is based are likely to be transferable to other nursing contexts.

Attitude of Health Personnel↗
PubMed2004

Ultraviolet-B signalling: Arabidopsis brassinosteroid mutants are defective in UV-B regulated defence gene expression.

The involvement of brassinosteroids in signalling events in plants during UV-B stress (280-315 nm) was investigated in Arabidopsis thaliana. Brassinosteroids are involved in growth and development in plants and have also been shown to enhance stress tolerance. Three mutants deficient in the biosynthetic pathway of brassinolide (BL; det2, dim1 and cpd) and the BL insensitive mutant (bri1) were together with visible light irradiated with 3 or 9 h of UV-B radiation (biologically effective radiation normalised to 300 nm being 0.24 W m(-2)). Also, a small size control, irx1, and Columbia 0 (Col-0) wild-type plants were examined under identical conditions. Gene expression patterns were established for these mutants with a set of four molecular markers (the defence genes chalcone synthase (CHS), PYROA, pathogenesis-related protein PR-5, and a gene regulated by very low levels of UV-B, MEB5.2). Although the genes in the brassinodefective mutants were still induced by UV-B radiation, they all also showed reduced levels of mRNA transcripts compared with Col-0 and irx1. The bri1 and cpd were the mutants with lowest levels of molecular marker mRNA transcripts. The effects of impairment of brassinosteroid signalling also differed between the genes studied, indicating a need for a complete brassinosteroid pathway in UV-B signalling.

Arabidopsis↗
PubMed2002

Childhood Miller Fisher syndrome: case report and review of the literature.

Three years ago Ray Buncic and I were having a break from the lectures at an ophthalmology meeting. Ray told me of his earnest desire to provide Canadian ophthalmologists with a series of updates in pediatric ophthalmology. After a few days we were both back to the "busi-ness" of medicine, with little time to devote to such a project. Then, along came the COS Council on Continuing Professional Development (CPD) and the MaintCert program--a bold initiative to draw talent of Canadian ophthalmology into a program of continuing medical education (CME). One initiative was to use the Canadian Journal of Ophthalmology as a CME tool. This article by Eedy Mezer and Ray Buncic is highlighted as a Section 3 learning activity (self-assessment). I am delighted to have seen Ray bring forward his idea in this manner. The Canadian Journal of Ophthalmology has initiated this project in conjunction with the Council on CPD. There are a number of questions that relate to this article that can be answered on the COS Web site (http://www.eyesite.ca). Participants will be provided with an aggregate score and a certificate that can be printed to allow them to record this activity for Section 3 credits with the Royal College of Physicians and Surgeons of Canada.

Child, Preschool↗
PubMed2000

Reconceptualising the outcomes of Continuing Professional Development.

This paper considers the potential outcomes, both positive and negative, of continuing professional development from the perspectives of practitioners and managers. Following a consideration of the literature it draws upon data collected during a 3-year evaluation of the English National Board Framework and Higher Award to highlight divergent views and tensions within Continuing Professional Development (CPD). Considerable discrepancies between practitioners and managers emerge which raise a number of searching questions about the value each group accords to continuing professional development. Based on the data a new framework for conceptualizing the outcomes of CPD is presented which fundamentally undermines a quasi-market approach.

Attitude of Health Personnel↗
PubMed1992

Surgical aspects of chronic peritoneal dialysis in the neonate and infant under 1 year of age.

Since 1982 eight patients under 1 year of age with end-stage renal failure have been treated by chronic peritoneal dialysis (CPD) following insertion of an abdominal Tenckhoff catheter. We routinely perform a partial omentectomy now, and in males undertake bilateral exploration of the groins at the time of catheter insertion, with herniotomy or ligation of the patent processus vaginalis as required. Up to January 1990, 19 straight double-cuff catheters had been inserted with a total follow-up of 244.5 patient months. The median age at the initial catheter insertion was 14.6 weeks (range, 2 days to 11 months) and the median weight was 3.89 kg (range, 2.2 to 5.5). Peritonitis was the most common complication, with 46 episodes, representing one episode of peritonitis per 5.3 patient months on dialysis. The frequency of peritonitis has decreased in the last 6 months since all patients have been dialysed by two caregivers. The present rate of peritonitis is 1 episode per 10 patient months on dialysis. One patient has died of septicemia secondary to associated congenital abnormalities, one patient has regained renal function, and two patients have been transplanted, one successfully. Five patients are currently dialysing via their abdominal Tenckhoff catheters and awaiting transplantation. We conclude that neonates and infants under 1 year of age can be treated satisfactorily by CPD to enable successful preparation for transplantation later in childhood.

Catheters, Indwelling↗
PubMed2000

Diurnal illumination patterns affect the development of the chick eye.

Exposure to continuous illumination disrupts normal ocular development in young chicks, causing severe corneal flattening, shallow anterior chambers and progressive hyperopia ('constant light (CL) effects'). We have studied the minimum requirements of a diurnal light cycle to prevent CL effects. (1) Seven groups of 10 chicks were reared under a 0 (constant light, CL), or 1, 2, 3,4, 6, or 12/12 h (normal) light-dark cycles. It was found that CL effects were prevented if the dark period was 4 h or longer. Below 4 h, the effects were dose-dependent and inversely correlated with the amplitude of the Fourier component of illumination at 1 cycle per day (CPD). (2) Three groups of 20 chicks were exposed to 4 h of darkness distributed differently over 24 h to vary the amplitude of the Fourier component at 1 CPD. It was found that complete suppression of the CL effects required that the 4 h of darkness were given in one block and at the same time each day. Our results show that normal ocular development in the chick requires a minimum of 4 h darkness per day, provided at the same time of the day without interruption, and suggest that the light-dark cycle interacts with a linear or weakly nonlinear oscillating system.

Animals↗
PubMed2001

Invariance of long-term visual priming to scale, reflection, translation, and hemisphere.

The representation of shape mediating visual object priming was investigated. In two blocks of trials, subjects named images of common objects presented for 185 ms that were bandpass filtered, either at high (10 cpd) or at low (2 cpd) center frequency with a 1.5 octave bandwidth, and positioned either 5 degrees right or left of fixation. The second presentation of an image of a given object type could be filtered at the same or different band, be shown at the same or translated (and mirror reflected) position, and be the same exemplar as that in the first block or a same-name different-shaped exemplar (e.g. a different kind of chair). Second block reaction times (RTs) and error rates were markedly lower than they were on the first block, which, in the context of prior results, was indicative of strong priming. A change of exemplar in the second block resulted in a significant cost in RTs and error rates, indicating that a portion of the priming was visual and not just verbal or basic-level conceptual. However, a change in the spatial frequency (SF) content of the image had no effect on priming despite the dramatic difference it made in appearance of the objects. This invariance to SF changes was also preserved with centrally presented images in a second experiment. Priming was also invariant to a change in left-right position (and mirror orientation) of the image. The invariance over translation of such a large magnitude suggests that the locus of the representation mediating the priming is beyond an area that would be homologous to posterior TEO in the monkey. We conclude that this representation is insensitive to low level image variations (e.g. SF, precise position or orientation of features) that do not alter the basic part-structure of the object. Finally, recognition performance was unaffected by whether low or high bandpassed images were presented either in the left or right visual field, giving no support to the hypothesis of hemispheric differences in processing low and high spatial frequencies.

Analysis of Variance↗
PubMed2002

Visual function before and after the removal of bilateral congenital cataracts in adulthood.

Subject Peter Doyle (PD) had congenital bilateral cataracts removed at the age of 43. Pre-operatively PD's visual acuity was 20/80, with a resolution limit around 15 cpd, and he experienced monocular diplopia with high contrast stimuli. Post-operatively PD's visual acuity improved to approximately 20/40, with a resolution limit around 25 cpd. Using a variety of pre- and post-operative tests we have documented a wide range of neural adaptations to his limited and distorted visual input, and have found a limited amount of post-operative adaptation to his newly improved visual input. These results show that the human visual system is capable of significant adaptation to the particular optical input that is experienced.

Adaptation, Physiological↗
PubMed1998

Luminance contrast and spatial-frequency tuning of the transient-vergence system.

Vergence has transient components that are stimulated by brief presentations of stimuli at large disparities (up to several degrees). The question that we have addressed is what stimulus features are encoded by this system. A competition paradigm [Jones & Kerr, (1972)]. Vision Research, 12, 1425-1430) was used in which three gabors were presented. A single Gabor was presented to the fovea of one eye and two gabors, 2.5 deg to either side of the fovea, to the other; one of which, when paired with the single Gabor defined a convergent direction, the other a divergent direction. First we determined if increasing the luminance contrast of the Gabor pair whose disparity was opposite to the observer's response-bias direction (variable-contrast pair) relative to the remaining Gabor (reference) could alter the observer's response direction. Secondly, we determined if the contrast required for such a change in response was affected by the relative spatial frequency of the convergent and divergent Gabors. The reference Gabor was held at 2 cpd and the variable Gabor pair was varied between 5.6 and 0 (a gaussian) cpd. Results demonstrated that increasing the luminance contrast of the variable pair relative to the reference Gabor could alter the observer's response direction, even when the contrast of only one of the variable-pair Gabors was increased. The luminance contrast required for this change to occur was directly related to the spatial frequency of the variable pair over the entire frequency range tested. Vergence responses were preferentially made to lower spatial frequencies, even when a low spatial frequency was pared with a high one. We conclude that transient-vergence responses are not reduced by mixed contrasts (i.e. no contrast-paradox effect) and appear to be mediated by a system that employs a single lowpass sensitive channel.

Contrast Sensitivity↗
PubMed1998

The effect of spatial frequency adaptation on the latency of spatial contrast detection.

The effect of spatial frequency adaptation on detection response time was studied using 2-D Gabor functions as stimuli. On the basis of pilot studies, it was expected that reaction time to a given spatial frequency at a low contrast would increase following adaptation to that spatial frequency at a high contrast. Subjects were tested using 2-D Gabor functions that ranged in frequency from 25 to 24 cpd. Subjects' reaction times to the Gabor functions were measured prior to adaptation and after adaptation to a particular spatial frequency. The adapting spatial frequency was either 1, 2, 4, 8, 10, or 16 cpd. The test stimuli were 0.3, 0.5, or 0.7 log units above the unadapted threshold contrast. The subjects adapted to the high contrast test grating for 3 min (80% contrast) and reaction times were again measured in an adapt-test-readapt paradigm. The results showed the greatest increase in reaction time after adaptation when adapting and test spatial frequencies within an octave of the adapting spatial frequency also showed an increase in reaction time but to a lesser extent. Reaction times to gratings with spatial frequencies more distant from the adapting spatial frequency were not significantly affected by the adaptation. The results obtained resemble the tuning curves found for threshold data. Reaction times for stimuli at 0.5 and 0.7 log units above the unadapted threshold were affected less by adaptation than those at 0.3 log units above the unadapted threshold. These results were evaluated in terms of a shifting contrast gain mechanism which may account for both the spatial frequency specific effects of adaptation and the differences found for the different contrast test levels.

Adaptation, Ocular↗
PubMed1999

Visual completion of partly occluded grating in infants under 1 month of age.

Four groups of eight infants (3 weeks of age on average) were each habituated to one of four displays consisting of a grating of either low (0.4 cpd) or high (1.2 cpd) spatial frequency, whose central portion was covered up with a horizontal occluder which was either narrow (1.33 degrees) or broad (4.17 degrees). These habituation displays are referred to as LN (low spatial frequency grating and narrow occluder), LB (low and broad), HN (high and narrow), and HB (high and broad) displays. Posthabituation-test displays consisted of a complete grating (CG) of the same frequency as the habituated grating along with a separate grating (SG) whose central portion was replaced with a black gap of the same height as the occluder in the habituation displays. Infants habituated to the LN display looked significantly longer at the SG than the CG display during posthabituation-test trials. Infants habituated to the LB and HN displays looked at the CG and SG displays, almost equally. In contrast, infants habituated to the HB display looked longer at the CG than the SG display. These results show that infants under 1 month of age can perceive the continuation of the grating behind the occluder, and that their visual completion on habituation displays can be evoked according to the interaction between the spatial frequency of the grating and the occluder height.

Evoked Potentials, Visual↗
PubMed1999

Senescent changes in scotopic contrast sensitivity.

Scotopic contrast sensitivity functions (CSFs) were measured for 50 observers between the ages of 20 and 88 years. Using a maximum-likelihood, 2-alternative, temporal forced-choice threshold-estimation algorithm, scotopic CSFs were measured at 7 spatial frequencies ranging from 0.2 to 3.0 cpd, with mean retinal illuminance equated for observers at -0.85 log scotopic Trolands. For each stimulus condition, eight cycles of a horizontal sinusoidal grating were presented within +/- 1 S.D. of a 2-D Gaussian-spatial envelope and within a 1-s Gaussian-temporal envelope. Stimuli were centered on the nasal retina along the horizontal meridian 6 degrees from the fovea. Scotopic CSFs were found to be low-pass. Statistically significant age-related declines in contrast sensitivities were found for spatial frequencies at or below 1.2 cpd. There was also a statistically significant decrease in the high frequency cut-off with age (P < 0.01). An explanation of these results in terms of optical factors is rejected, while the results are consistent with age-related changes in the magnocellular pathway.

Adult↗
PubMed2000

Spatial resolution and receptive field height of motion sensors in human vision.

We estimated the length of motion-detecting receptive fields in human vision by measuring direction discrimination for three novel stimuli. The motion sequences contained either (i) alternate frames of two differently oriented sinusoidal gratings; (ii) alternate frames of vertical grating and plaid stimuli or (iii) a vertical grating divided into horizontal strips of equal height, where alternate strips moved leftward and rightward. All three stimulus sequences had a similar appearance (of moving strips) and the task was to identify the direction of the central strip. For sequences (ii) and (iii), performance fell as the strip height decreased. Threshold height fell with increasing contrast up to about 20%, then levelled off at the critical strip height. Temporal frequency (1. 9-15 Hz) had no effect on the critical strip height. We argue that the receptive field length corresponds to twice this critical height. The length estimates were strikingly short, ranging from about 0.4 cycles at 3.0 cpd to 0.1 cycles at 0.1 cpd. These lengths agree well with the estimates derived at threshold by Anderson and Burr (1991, J. Opt. Soc. Am. A8, 1330-1339), and imply that the motion-sensing filters have very broad orientation tuning. These and other results are interpreted within the framework of a Gaussian derivative model for motion filtering. The sensitivity of motion filters to a broad range of orientations suggests a simpler view of how coherent plaid motion is processed.

Humans↗
PubMed2005

Plant brassinosteroid hormones.

In animals, a large number of steroid hormones play important roles in numerous processes including reproduction and differentiation. The biologically active plant steroid brassinolide (BL) was first discovered in the pollen of western rape in 1979 (Grove et al., 1979). This finding suggested that BL is indispensable for plant growth and differentiation. To date, more than 50 BL analogs have been identified, and the group has been termed brassinosteroids (BRs) (Fujioka and Yokota, 2003). Brassinosteroids have several biological activities, such as inducing cell elongation when applied at very low concentrations. For this reason, soon after their discovery, they were suggested to be a sixth type of plant hormone; however, for years BRs were not considered true plant hormones. The turning point in BR research was the discovery of the Arabidopsis dwarf mutants det2 and cpd in 1996 (Li et al., 1996; Szekeres et al., 1996). These BR-deficient mutants were found to revert to the wild-type phenotype following BR treatment. Concurrent with the analysis of these mutants, an outline of the biosynthetic pathway of BRs was being elucidated through chemical analysis. Following the isolation of det2 and cpd, a great number of BR-deficient mutants were identified. The mutant genes were found to encode proteins that catalyze the conversion of plant steroids to BR precursors. Eventually, BRs were widely recognized as important plant hormones indispensable for growth and differentiation (Clouse and Sasse, 1998). In parallel, mutants that are insensitive to BRs were isolated (Clouse et al., 1996; Li et al., 1997) with phenotypes very similar to those of the BR-biosynthesis mutants. Investigations of these mutants revealed several mechanisms of BR perception and signal transduction (Bishop and Koncz, 2002; Clouse, 2002). This review describes findings on the effects of BRs on plant growth, BR biosynthesis and catabolism, and BR signal transduction.

Brassinosteroids↗
PubMed1998

Biochemical characterization of a haloalcohol dehalogenase from Arthrobacter erithii H10a.

Arthrobacter erithii H10a possesses two enzymes capable of catalyzing the dehalogenation of vicinal halohydrins which have been designated as dehalogenases DehA and DehC. The DehA dehalogenase demonstrated greater activity toward 1,3-dichloro-2-propanol (1,3-DCP) while the DehC dehalogenase showed higher activity toward 3-chloro-1,2-propanediol (3-CPD) and brominated alcohols. The DehA dehalogenase was composed of two non-identical subunits (relative molecular mass of 31.5 and 34 kDa) which probably associate with other proteins to form a large catalytically active protein of 200 kDa. The two subunits were purified and the amino acid sequence of their tryptic digests determined. The DehA enzyme catalyzed the conversion of vicinal halohydrins to epoxides and the reverse reaction in the presence of an excess of halogen. This enzyme had maximum activity at 50 degrees C and a broad pH optimum over the range 8.5-10.5. The apparent K(m) and Vmax values for dehalogenation of 1,3-DCP and 3-CPD were 0.105 mM and 223 mumol min-1 mg-1; and 2.366 mM and 1.742 mumol min-1 mg-1, respectively. The enzyme was inhibited by 2-chloroacetic acid (MCA) and 2,2-dichloroacetic acid (DCA). The inhibition pattern suggested a mixed type inhibition which was predominantly uncompetitive. Amino acid modification experiments demonstrated that one or more cysteine and arginine residues are likely to be involved in catalysis or play an important role in the maintenance of the enzyme structure. The characteristics of the DehA enzyme are compared to those of previously reported haloalcohol dehalogenases and discussed in terms of diversity of this type of dehalogenase.

Alcohols↗