Sculpting the proteome with AAA(+) proteases and disassembly machines

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.     

Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae

When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We found that the diauxic shift causes no overall change in translational accuracy but does cause a strong reduction in the frequency of one type of programmed error: Ty +1 frameshifting. Genetic data suggest that this effect may be due to changes in the relative amounts of tRNA participating in translation elongation. We discuss possible implications for expression strategies that use recoding.     

High-performance peptide identification by tandem mass spectrometry allows reliable automatic data processing in proteomics

In a previous paper we introduced a novel model-based approach (OLAV) to the problem of identifying peptides via tandem mass spectrometry, for which early implementations showed promising performance. We recently further improved this performance to a remarkable level (1-2% false positive rate at 95% true positive rate) and characterized key properties of OLAV like robustness and training set size. We present these results in a synthetic and coherent way along with detailed performance comparisons, a new scoring component making use of peptide amino acidic composition, and new developments like automatic parameter learning. Finally, we discuss the impact of OLAV on the automation of proteomics projects.     

Individual differences in respiratory sinus arrhythmia

To investigate the interindividual differences in respiratory sinus arrhythmia (RSA), recordings of ventilation and electrocardiogram were obtained from 12 healthy subjects for five imposed breathing periods (T(TOT)) surrounding each individual's spontaneous breathing period. In addition to the spectral analysis of the R-R interval signal at each breathing period, RSA characteristics were quantified by using a breath-by-breath analysis where a sinusoid was fitted to the changes in instantaneous heart rate in each breath. The amplitude and phase (or delay = phase x T(TOT)) of this sinusoid were taken as the RSA characteristics for each breath. It was found that for each subject the RSA amplitude-T(TOT) relationship was linear, whereas the delay-T(TOT) relationship was parabolic. However, the parameters of these relationships differed between individuals. Linear correlation between the slopes of RSA amplitude versus T(TOT) regression lines and 1) mean breathing period and 2) mean R-R interval during spontaneous breathing were calculated. Only the correlation coefficient with breathing period was significantly different from zero, indicating that the longer the spontaneous breathing period the lesser the increase in RSA amplitude with increasing breathing period. Similarly, only the correlation coefficient between the curvature of the RSA delay-T(TOT) parabola and mean breathing period was significantly different from zero; the longer the spontaneous breathing period the larger the curvature of RSA delay. These results suggest that the changes in RSA characteristics induced by changing the breathing period may be explained partly by the spontaneous breathing period of each individual. Furthermore, a transfer function analysis performed on these data suggested interindividual differences in the autonomic modulation of the heart rate.     

Psychosocial effects of SARS on hospital staff: survey of a large tertiary care institution

BackgroundThe outbreak of SARS in 2003 had a dramatic effect on the health care system in Toronto. The main objective of this study was to investigate the psychosocial effects associated with working in a hospital environment during this outbreak.MethodsQuestionnaires were distributed to all willing employees of Sunnybrook and Women''s College Health Sciences Centre between Apr. 10 and 22, 2003. The survey included questions regarding concern about SARS, precautionary measures, personal well-being and sociodemographic characteristics; a subsample also received the 12-item version of the General Health Questionnaire (GHQ-12).ResultsOf the 4283 questionnaires distributed, 2001 (47%) were returned, representing 27% of the total hospital employee population of 7474. The proportions of respondents who were allied health care professionals, nurses and doctors and who worked in areas other than patient care were representative of the hospital staff population as a whole. Of the 2001 questionnaires, 510 contained the GHQ-12. Two-thirds of the respondents reported SARS-related concern for their own or their family''s health. A total of 148 respondents (29%) scored above the threshold point on the GHQ-12, indicating probable emotional distress; the rate among nurses was 45%. Masks were reported to be the most bothersome infection control precaution. Logistic regression analysis identified 4 factors as being significantly associated with increased levels of concern for personal or family health: perception of a greater risk of death from SARS (adjusted odds ratio [OR] 5.0, 95% confidence interval [CI] 2.6–9.6), living with children (adjusted OR 1.8, 95% CI 1.5–2.3), personal or family lifestyle affected by SARS outbreak (adjusted OR 3.3, 95% CI 2.5–4.3) and being treated differently by people because of working in a hospital (adjusted OR 1.6, 95% CI 1.2–2.1). Four factors were identified as being significantly associated with the presence of emotional distress: being a nurse (adjusted OR 2.8, 95% CI 1.5–5.5), part-time employment status (adjusted OR 2.6, 95% CI 1.2–5.4), lifestyle affected by SARS outbreak (adjusted OR 2.2, 95% CI 1.4–3.5) and ability to do one''s job affected by the precautionary measures (adjusted OR 2.9, 95% CI 1.9–4.6).InterpretationOur findings indicate that the SARS outbreak had significant psychosocial effects on hospital staff. These effects differed with respect to occupation and risk perception. The effect on families and lifestyle was also substantial. These findings highlight the need for interventions to address psychosocial distress and concern and to provide support for employees during such crises.During the spring of 2003, Toronto was in the midst of the first of 2 phases of a SARS outbreak. As the principal tertiary referral hospital, Sunnybrook and Women''s College Health Sciences Centre (SWC) admitted 71 patients with SARS, of whom 23 were health care workers, between Mar. 14 and May 24. Over 1000 patients were seen at the SWC SARS assessment clinic.The effect of SARS on the health care system in the greater Toronto area was dramatic.^1,2,3,4,5,6 At various times during the outbreak, 3 hospitals were closed. Health care workers were at increased risk and many were quarantined, which resulted in severe staff shortages. On Mar. 28, following the closure of a second hospital, new and intensive infection control directives were issued for all hospitals in the greater Toronto area and surrounding area. At SWC the directives included cancellation of all hospital-based outpatient clinics, significant visitor restrictions, mandatory wearing of surgical masks by all staff at all times (and N95 masks in patient care areas), limited hospital entrance and mandatory screening of everyone entering the building (symptom/exposure questionnaire and temperature reading). Health care workers were instructed to work at 1 health care institution only, and off-work contact between health care workers from different institutions was discouraged. The SWC SARS Management Team met daily to implement Ministry of Health directives, organize care of patients with and without SARS and deal with staffing issues. With clinic and operating room closures and quarantined staff, staff redeployment to screening at entrances and other essential services became necessary. After Apr. 17, staff not involved in patient care no longer had to wear masks; however, most of the other infection control directives were kept in place well into the summer months.Little is known about the psychological effects of this type of disease outbreak on health care and other hospital workers. Maunder and colleagues¹ described the experiences of a small number of patients and staff at a Toronto hospital during the initial SARS outbreak. They observed that the staff were fearful for their own and their family''s health and found caring for colleagues as patients emotionally difficult. Mitchell and associates⁷ reported increased feelings of stigmatization among nurses during an outbreak of vancomycin-resistant enterococci in a hospital in Australia; feelings of alienation and isolation were also noted. A literature review revealed no large, systematic studies of the effect of a disease outbreak on hospital staff, particularly in cases with a high risk of nosocomial spread, as is the case with SARS.^2,4,8,9,10The main objectives of this study were (a) to determine the self-reported psychosocial effects associated with working in a hospital environment during the peak of a disease outbreak, specifically psychological distress and effects on the work and personal lives of employees, and (b) to examine the determinants of these effects.     

CXCL‐10 and Interleukin‐6 are reliable serum markers for vitiligo activity: A multicenter cross‐sectional study

This cross‐sectional multicenter study aimed to evaluate serum CXCL‐10, as an activity marker for vitiligo, and compare it with other putative serum and tissue markers. Serum CXCL‐10 was compared to interferon gamma (IFN‐γ), interleukin 6 (IL‐6), and IL‐17 using ELISA in 55 non‐segmental vitiligo patients (30 active and 25 stable) and 30 healthy controls. Marginal skin biopsy was taken for immunohistochemical evaluation of CD8+T cells and CXCL‐10+ve cells. Serum levels of CXCL‐10, IL‐17, and IL‐6 were elevated in all vitiligo patients compared to controls (p < .05). All investigated serum markers were higher in active versus stable vitiligo. Tissue expression of CXCL‐10+ve cells and CD8+ve T cells was stronger in vitiligo patients compared to controls, and tissue CXCL‐10+ve cell expression was stronger in active versus stable cases. Positive correlations were noted between the different serum and tissue markers. CXCL‐10 was the most specific, whereas IL‐6 was the most sensitive serum marker to distinguish active from stable disease.     

Biosynthesis of phenolic compounds inVitis vinifera cell suspension cultures: Study on hydroxycinnamoyl CoA:ligase

In cell suspensions cultures from grape berry pulp (Vitis vinifera cv. Gamay fréaux)hydroxycinnamoyl CoA ligase (CoAL) displayed maximum activity (100 %) forp-coumaric acid and then, in decreasing order, for ferulic acid (81.3 %) and caffeic acid (60.4 %). No activity was detected with sinapic and cinnamic acids. The changes in CoAL activity during the growth cycle of the culture displayed two peaks : the highest (6 h after subculturing) was linked with a strong increase in protein caused by dilution ; the second was weaker and occurred on the 7th day of culture.Grape cell suspension accumulated mainly peonidin (Pn) and cyanidin (Cy) glucosides (Pn 3-glucoside, Cy 3-glucoside, Pn 3-acetylglucoside, Pn 3-caffeylglucoside, Pn 3-p-coumarylglucoside, and Cy 3-p-coumarylglucoside). Maximum accumulation of anthocyanins was associated with the exponential growth phase of the culture and might be the result of the substantial increase in CoAL activity resulting from the effect of dilution. The second enzyme activity peak was probably oriented towards the acylation of anthocyanins since the percentage of acylated forms increased with time after subculturing.Abbreviations CoAL hydroxycinnamoyl-CoA: ligase     

Desipramine Elicits the Expression of Opiate Receptors and Sulfogalactosylceramide Synthesis in Rat C6 Glioma Cells

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.     

Ventilatory conditioning by self-stimulation in rats: A pilot study

This article describes an experimental attempt to condition breathing pattern in rats. In this experiment, a freely moving rat was first rewarded by an electrical stimulation of the medial forebrain bundle whenever inspiratory duration (TI) exceeded 300 ms. A bidirectional control was then used: TIs longer than 400 ms were rewarded, and then TIs shorter than 300 ms were rewarded. The frequency of TIs longer than 300 ms increased when this event was rewarded, further increased when TIs above 400 ms were rewarded, and decreased during reversal conditioning (TI < 300 ms). At the beginning of the experiment, stimulation caused increased arousal and motor activity, but after prolonged conditioning, the brain stimulation was associated with quiet wakefulness. Although the general procedure appears to be well-suited to the experimental study of voluntary breathing, some possible improvements are suggested for further, more extensive investigations.We are grateful to Professor Claude Gaultier and to Professor Vincent Bloch for their support, to Pascale Leblanc for her invaluable assistance, and to Gerard Dutrieux and Michel Vigouroux for their technical contribution. This work was supported by the Université de Paris-Sud (Grant AI-9023) and by the Institut National de la Sante et de la Recherche Médicale (CJF 89-09).