Newly developed waist actigraphy and its sleep/wake scoring algorithm

The purpose of this study was to formulate an algorithm for assessing sleep/waking from activity intensities measured with a waist-worn actigraphy, the Lifecorder PLUS (LC; Suzuken Co. Ltd., Nagoya, Japan), and to test the validity of the algorithm. The study consisted of 31 healthy subjects (M/F = 20/11, mean age 31.7 years) who underwent one night of simultaneous measurement of activity intensity by LC and polysomnography (PSG). A sleep(S)/wake(W) scoring algorithm based on a linear model was determined through discriminant analysis of activity intensities measured by LC over a total of 235 h and 56 min and the corresponding PSG-based S/W data. The formulated S/W scoring algorithm was then used to score S/W during the monitoring epochs (2 min each, 7078 epochs in total) for each subject. The mean agreement rate with the corresponding PSG-based S/W data was 86.9%, with a mean sensitivity (sleep detection) of 89.4% and mean specificity (wakefulness detection) of 58.2%. The agreement rates for the individual stages of sleep were 60.6% for Stage 1, 89.3% for Stage 2, 99.2% for Stage 3 + 4, and 90.1% for Stage REM. These results demonstrate that sleep/wake activity in young to middle-aged healthy subjects can be assessed with a reliability comparable to that of conventional actigraphy through LC waist actigraphy and the optimal S/W scoring algorithm.

     

Validation of the Chinese Version of the Children’s ChronoType Questionnaire (CCTQ) in school-aged children

ABSTRACT

The Children’s ChronoType Questionnaire (CCTQ) is a valid and reliable measure for assessing prepubertal children aged 4–11 years. The CCTQ is a parent-reported, 27-item questionnaire consisting of sleep-wake parameters for scheduled and free days (16 items), a morningness/eveningness scale (M/E, 10 items), and a five-point, single-item, chronotype score. The CCTQ has been translated into different languages, but a Chinese version is not available. In the present study, we aimed to produce a Chinese version of the CCTQ and test its validity and reliability on school-aged children. A total of 555 children aged 7–11 years were recruited from five primary schools. The parents were told to complete the CCTQ and record their child’s sleep pattern in a 7-day sleep diary. Sixty-six children and their parents were invited to participate in determining the test-retest reliability of the CCTQ over a 2-week interval, and their sleep patterns were assessed using a sleep diary. The internal consistency of the Chinese CCTQ M/E score as measured by Cronbach’s alpha was acceptable (0.74). Regarding the test-retest reliability of the instrument, moderate to strong Spearman’s correlation coefficients were found for most of the CCTQ – sleep-wake items (ρ = 0.52–0.86) and for the CCTQ-M/E total score (ρ = 0.78). For the concurrent validity, Spearman’s correlations between the sleep-wake parameters of the CCTQ and the sleep diary were moderate to high on both the scheduled days (ρ = 0.54 to 0.87) and free days (ρ = 0.36 to 0.60). For the correlations measured with actigraphs, significant correlations were found in the CCTQ sleep-wake parameters, including bedtime, get-up time, sleep latency, sleep period, time in bed, and mid-sleep point on both the scheduled (ρ = 0.31 to 0.76) and free days (ρ = 0.27 to 0.52), but not in sleep latency and sleep period on free days. The results of the present study suggest that the Chinese version of the CCTQ is a reliable and valid tool for assessing chronotypes in Chinese school-aged children in Hong Kong.     

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).     

Heritability of Morningness‐Eveningness and Self‐Report Sleep Measures in a Family‐Based Sample of 521 Hutterites

Individual variation in the phase and amplitude of human circadian rhythms is well known, but the impact of heritable factors on such variation is less clear. We estimated the narrow‐sense heritability for selected circadian and sleep timing, quality, and duration measures among related members of the Hutterites, an endogamous, religious community (n=521 participants). “Morningness‐eveningness” (M/E), a stable trait reflecting circadian phase, was evaluated using the Composite Scale (CS). Subjective sleep measures were assessed using the Sleep Timing Questionnaire. Initial analyses reconfirmed the impact of age on M/E. Previously reported correlations between M/E scores and the sleep measures were also noted, demonstrating the construct validity of the questionnaires among the participants. Following corrections for age, gender, and colony of residence, significant narrow‐sense heritability was noted for M/E (23%). The heritability for subjective sleep measures (related to timing, duration, and quality) were statistically significant for all but one variable, and varied between 12.4% and 29.4%. Thus, significant heritable influences on human circadian phase and subjective sleep indices can be detected through family‐based studies. In view of the impact of circadian malfunction on human health, it may be worthwhile to map genetic factors impacting circadian and sleep variation.     

Heritability of morningness-eveningness and self-report sleep measures in a family-based sample of 521 hutterites

Individual variation in the phase and amplitude of human circadian rhythms is well known, but the impact of heritable factors on such variation is less clear. We estimated the narrow-sense heritability for selected circadian and sleep timing, quality, and duration measures among related members of the Hutterites, an endogamous, religious community (n=521 participants). “Morningness-eveningness” (M/E), a stable trait reflecting circadian phase, was evaluated using the Composite Scale (CS). Subjective sleep measures were assessed using the Sleep Timing Questionnaire. Initial analyses reconfirmed the impact of age on M/E. Previously reported correlations between M/E scores and the sleep measures were also noted, demonstrating the construct validity of the questionnaires among the participants. Following corrections for age, gender, and colony of residence, significant narrow-sense heritability was noted for M/E (23%). The heritability for subjective sleep measures (related to timing, duration, and quality) were statistically significant for all but one variable, and varied between 12.4% and 29.4%. Thus, significant heritable influences on human circadian phase and subjective sleep indices can be detected through family-based studies. In view of the impact of circadian malfunction on human health, it may be worthwhile to map genetic factors impacting circadian and sleep variation.     

Sleep and eating behavior in adults at risk for type 2 diabetes

Insufficient quantity and quality of sleep may modulate eating behavior, everyday physical activity, overall energy balance, and individual risk of obesity and type 2 diabetes. We examined the association of habitual sleep quantity and quality with the self-reported pattern of eating behavior in 53 healthy urban adults with parental history of type 2 diabetes (30 F/23 M; mean (s.d.) age: 27 (4) years; BMI: 23.9 (2.3) kg/m(2)) while taking into consideration the amount of their everyday physical activity. Participants completed 13 (3) days of sleep and physical activity monitoring by wrist actigraphy and waist accelerometry while following their usual lifestyle at home. Overnight laboratory polysomnography was used to screen for sleep disorders. Subjective sleep quality was measured with the Pittsburgh Sleep Quality Index. Eating behavior was assessed using the original 51-item and the revised 18-item version of the Three-Factor Eating Questionnaire including measures of cognitive restraint, disinhibition, hunger, and uncontrolled and emotional eating. In multivariable regression analyses adjusted for age, BMI, gender, race/ethnicity, level of education, habitual sleep time measured by wrist actigraphy and physical activity measured by waist accelerometry, lower subjective sleep quality was associated with increased hunger, more disinhibited, uncontrolled and emotional eating, and higher cognitive restraint. There was no significant association between the amount of sleep measured by wrist actigraphy and any of these eating behavior factors. Our findings indicate that small decrements in self-reported sleep quality can be a sensitive indicator for the presence of potentially problematic eating patterns in healthy urban adults with familial risk for type 2 diabetes.     

A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Nav1.7 channel is disrupted by the erythromelalgia-associated F1449V mutation

Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.     

The reliability and validity of the Japanese version of the Children's ChronoType Questionnaire (CCTQ) in preschool children

We aimed to examine the reliability and validity of the Japanese version of the Children's ChronoType Questionnaire (CCTQ) in preschool children. The CCTQ consists of 16 items on sleep–wake parameters for scheduled and free days, a 10-item of the Morningness/Eveningness Scale (CCTQ-M/E), and a single item on chronotype. Out of 502 children aged 3–6 years living in Okayama Prefecture, we evaluated 346 (188 boys and 158 girls) between May and June 2012. Their parents filled out the questionnaires two times at an interval of two weeks. Cronbach's α of the CCTQ-M/E was 0.77. For test–retest reliability, Pearson's correlation coefficient of the CCTQ-M/E between the two observations was 0.898 (p?<?0.001). Kruskal–Wallis test with post-hoc tests was used to compare sleep–wake parameters measured with the CCTQ among the three groups of children, morning (M)-type, neither (N)-type and evening (E)-type, who were classified according to the CCTQ-M/E score. Sleep–wake parameters in timing were significantly different among the children with M-type, N-type and E-type (p?<?0.001). Post-hoc pairwise comparisons revealed that sleep–wake parameters in timing were significantly delayed from the M-type to the N-type children (p?<?0.001), from the M-type to the E-type children (p?<?0.001), and from the N-type to the E-type children (p?<?0.001), except that wake-up time and get-up time were not significantly different between the children with N-type and E-type on scheduled days when their start time was regularly fixed. Out of these 346 children, we evaluated 72 (35 boys and 37 girls) to see the correlations between subjectively and objectively measured sleep–wake parameters from June to October 2012. Spearman's correlation coefficients between sleep–wake parameters measured with the CCTQ and an actigraph were 0.512–0.836 on scheduled days (p?<?0.001) and 0.380–0.786 on free days (p?<?0.001). Based on these findings we conclude that the Japanese version of the CCTQ is a reliable and valid measure for assessing chronotypes in preschool children.     

A psychometric investigation of the sleep,circadian rhythms,and mood (SCRAM) questionnaire

The sleep, circadian rhythms, and mood (SCRAM) questionnaire (Byrne, Bullock et al., 2017) was designed to concurrently measure individual differences in three clinically important functions: diurnal preference, sleep quality, and mood. The 15-item questionnaire consists of three 5-item scales named Morningness, Good Sleep, and Depressed Mood. The overarching aim of the current project was to investigate the validity and reliability of the questionnaire. Here, we report on associations investigated in three data sets. Study 1 (N = 70, 80% females) was used to examine the test–retest reliability of the questionnaire, finding strong test–retest reliability of the three scales over a 2-week period (r’s ranging from 0.73 to 0.86). Study 2 (N = 183, 80% females) enabled us to examine the construct validity of the SCRAM scales against well-validated self-report measures of diurnal preference, sleep quality, and depression. Strong correlations were found between each SCRAM scale and their respective measure in bivariate analyses, and associations were robust after the inclusion of the remaining two SCRAM scales as predictors in regression analyses. Data from Study 3 (N = 42, 100% males) were used to measure the extent to which SCRAM scores correlated with objective measures of sleep–wake behavior using actigraphy. Morningness was found to be related to earlier sleep onset and offset times, and Good Sleep was related to higher sleep efficiency but to no other measures of sleep quality; Depressed Mood was not related to actigraphy measures. The findings provide provisional support for construct validity and reliability of the SCRAM questionnaire as a measure of diurnal preference, sleep quality, and depressed mood. Future research into the psychometrics of SCRAM should test the questionnaire’s discriminant and predictive validity in clinical samples.     

Empirical evaluation of a model-driven approach to enlargement of multi-dimensional questionnaires for assessing adaptability of the sleep–wake cycle

The spherical cube model was earlier proposed for explaining interrelationships between scales of multidimensional questionnaires designed for assessing adaptability of the human sleep–wake cycle. The purpose of this report was to use the model’s predictions for identification of new items associated with yet unassessed sub-traits of the sleep–wake adaptability. The 72-item Sleep–Wake Pattern Assessment Questionnaire (SWPAQ) and an initial 320-item list created for a new inventory were administered to 139 respondents. Results of correlating the responses to these items with scores on six SWPAQ’ scales were used for classification of items in accord with the nomenclature proposed by the model and for selection of 120 items for the new inventory that allowed the assessment of the majority (more than 24) of the sleep–wake adaptability sub-traits predicted by the model. Some of these newly assessed sub-traits reflect individual variation in the success of biological adaptation to night and shift work.     

Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups

The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [(3)H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.     

Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552: reciprocal mutation analysis

Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.     

Structure-activity studies of the inhibition of FabI,the enoyl reductase from Escherichia coli,by triclosan: kinetic analysis of mutant FabIs

Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.     

A multi-modal questionnaire for stress

This paper presents data regarding the Strain Questionnaire (SQ), a 48-item scale designed to measure self-report levels of behavioral, cognitive, and physical stress complaints. A heterogeneous sample of 412 adults completed the SQ, and correlational, reliability, factor analytic and validity data are reported. Results suggest that the items reflect a wide range of content which show high internal consistency on each of the three subscales and the total SQ. Test-retest reliability demonstrated satisfactory temporal stability of the scales. Factor analysis of the SQ produced eleven orthogonal factors. The most salient factor was "cognitive/behavioral strain" which highlights the necessity for including these variables in self-report surveys of stress. Future research needs are also discussed.     

Mood, alertness, and performance in response to sleep deprivation and recovery sleep in experienced shiftworkers versus non-shiftworkers

Previous studies have shown increased sleepiness and mood changes in shiftworkers, which may be due to sleep deprivation or circadian disruption. Few studies, however, have compared responses of experienced shiftworkers and non-shiftworkers to sleep deprivation in an identical laboratory setting. The aim of this laboratory study, therefore, was to compare long-term shiftworkers and non-shiftworkers and to investigate the effects of one night of total sleep deprivation (30.5 h of continuous wakefulness) and recovery sleep on psychomotor vigilance, self-rated alertness, and mood. Eleven experienced male shiftworkers (shiftwork ≥5 yrs) were matched with 14 non-shiftworkers for age (mean ± SD: 35.7 ± 7.2 and 32.5 ± 6.2 yrs, respectively) and body mass index (BMI) (28.7 ± 3.8 and 26.6 ± 3.4 kg/m(2), respectively). After keeping a 7-d self-selected sleep/wake cycle (7.5/8 h nocturnal sleep), both groups entered a laboratory session consisting of a night of adaptation sleep and a baseline sleep (each 7.5/8 h), a sleep deprivation night, and recovery sleep (4-h nap plus 7.5/8 h nighttime sleep). Subjective alertness and mood were assessed with the Karolinska Sleepiness Scale (KSS) and 9-digit rating scales, and vigilance was measured by the visual psychomotor vigilance test (PVT). A mixed-model regression analysis was carried out on data collected every hour during the sleep deprivation night and on all days (except for the adaptation day), at .25, 4.25, 5.25, 11.5, 12.5, and 13.5 h after habitual wake-up time. Despite similar circadian phase (melatonin onset), demographics, food intake, body posture, and environmental light, shiftworkers felt significantly more alert, more cheerful, more elated, and calmer than non-shiftworkers throughout the laboratory study. In addition, shiftworkers showed a faster median reaction time (RT) compared to non-shiftworkers, although four other PVT parameters did not differ between the groups. As expected, both groups showed a decrease in subjective alertness and PVT performance during and following the sleep deprivation night. Subjective sleepiness and most aspects of PVT performance returned to baseline levels after a nap and recovery sleep. The mechanisms underlying the observed differences between shiftworkers and non-shiftworkers require further study, but may be related to the absence of shiftwork the week prior to and during the laboratory study as well as selection into and out of shiftwork.     

Phase relationships between sleep-wake cycle and underlying circadian rhythms in Morningness-Eveningness

A shorter phase angle between habitual wake time and underlying circadian rhythms has been reported in evening types (E types) compared to morning-types (M types). In this study, phase angles were compared between 12 E types and 12 M types to verify if this difference was observed when the sleep schedule was relatively free from external social constraints. Subjects were selected according to their Morningness-Eveningness Questionnaire score (MEQ score). There were 6 men and 6 women in each group (ages 19-34 years), and all had a habitual sleep duration between 7 and 9 h. Sleep schedule was recorded by actigraphy and averaged over 7 days. Circadian phase was estimated by the hour of temperature minimum (T(min)) in a 26-h recording and by the timing of the onset of melatonin secretion (dim-light melatonin onset [DLMO]) measured in saliva samples. Phase angles were defined as the interval between phase markers and averaged wake time. Results showed that, in the present experimental conditions, phase angles were very similar in the 2 groups of subjects. However, results confirmed the previously reported correlation between phase and phase angle, showing that a later circadian phase was associated with a shorter phase angle. Gender comparisons showed that for a same MEQ score, women had an earlier DLMO and a longer phase angle between DLMO and wake time. Despite a significant difference in the averaged circadian phases between E-type and M-type groups, there was an overlap in the circadian phases of the subjects of the 2 groups. Further comparisons were made between the 2 circadian types, separately for the subgroups with overlapping or nonoverlapping circadian phases. In both subgroups, the significant difference between MEQ scores, bedtimes, and wake times were maintained in the expected direction. In the subgroup with nonoverlapping circadian phases, phase angles were shorter in E-type subjects, in accordance with previous studies. However, in the overlapping subgroup, phase angles were significantly longer in E types compared to M types. Results suggest that the morningness-eveningness preference identified by the MEQ score refers to 2 distinct mechanisms, 1 associated with a difference in circadian period and phase of entrainment and the other associated with chronobiological aspects of sleep regulation.     

Early side effects of intrathoracic BCG therapy in patients with stage I squamous cell carcinoma,adenocarcinoma, or large cell lung cancer

Summary Data are presented on 179 stage I lung cancer patients subjected to resection operations and then given adjuvant intrapleural BCG and subsequent isoniazid (INH) therapy and on 167 control patients given intrapleural saline and placebo pills in a two-arm randomized double-blind study. The predominant immediate response to BCG/INH therapy was hyperpyrexia, which was found to be more pronounced in patients with larger induration in pretreatment PPD skin tests. Subsequently, chemical hepatitis (6 cases after BCG/INH versus 1 case after saline/placebo), peripheral neuropathy (3 versus 1), dermatitis/hives (5 versus 2), pleural thickening (4 versus 0), and persistent fever (10 versus 0) were noted. Analysis of laboratory changes measured at 18 weeks following randomization revealed that patients with BCG/INH lost 1.1 kg in weight and 0.30 g/dl in hemoglobin concentration on average, whereas control patients gained 1.2 kg and 0.33 g/dl, respectively. Modest rises in SGOT and alkaline phosphatase were apparent at 6 weeks after instillation of BCG compared with controls, but these differences were no longer statistically signifikant after 18 weeks. These side effects notwhithstanding, the BCG/INH therapy was well tolerated.Members of the Lung Cancer Study Group include R. T. Eagan*, R. E. Lee, W. S. Payne, R. E. Ritts, and L. Weiland from the Mayo Clinic, Rochester; C. F. Mountain*, H. T. Barkley, O. H. Frazier, K. Hermes, E. Hersh, and M. Valdivieso from M.D. Anderson Hospital, Houston; L. D. Hill*, M. D. Hafermann, and E. Morgan from The Mason Clinic, Seattle; P. W. Wright* and K.-E. Hellstrom from the Hutchinson Cancer Center, Seattle; C. Bagley, L. P. Johnson, H. Kellogg, and R. D. Pinkham from the Swedish Medical Center, Seattle; T. D. Ivey from University Hospital, Seattle; S. Hammar from Virginia Mason Hospital, Seattle; W. Nelems from St. Paul's Hospital, Vancouver; R. Feld*, D. Bergsagel, T. C. Brown, J. Curtis, C. Keen, J. F. Pringle, I. Quirt, and L. Yeoh from The Princess Margaret Hospital, Toronto; M. Blackstein and M. Goldberg from Mount Sinai Hospital, Toronto; F. G. Pearson*, D. W. Chamberlain, J. Cooper, W. Evans, and T. Todd from Toronto General Hospital, Toronto; M. Baker and R. Ginsberg from Toronto Western Hospital, Toronto; R. I. Mitchell from Wellesley Hospital, Toronto; E. C. Holmes*, W. F. Coulson, K. P. Ramming, and T. H. Weisenburger from the University of California, Los Angeles; Z. Petrovich from Wadsworth Veterans Hospital, Los Angeles; R. K. Oldham*, J. T. Forbes, F. A. Greco, D. L. Page, R. Prager, R. L. Richardson, and S. L. Stroup from Vanderbilt University, Nashville; J. M. Lukeman* and S. M. Sajjad from the Pathology Reference Center of M.D. Anderson Hospital, Houston; P. Grifone, A. Lebeck, and T. Voss from the Operations Office, Silver Spring, Maryland; M. Gail, W. McGuire, J. Allegra, and L. Rubinstein from the National Cancer Institute, Bethesda, Maryland; and L. Eirich, W. Heineman, and J. Beach from Information Management Services, Bethesda, Maryland. Asterisks designate principal investigators.     

Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire

The Agrobacterium sp. beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.     

Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2

Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.