A minimal system for Tn7 transposition: the transposon-encoded proteins TnsA and TnsB can execute DNA breakage and joining reactions that generate circularized Tn7 species.

In the presence of ATP and Mg(2+), the bacterial transposon Tn7 translocates via a cut and paste mechanism executed by the transposon-encoded proteins TnsA+TnsB+TnsC+TnsD. We report here that in the presence of Mn(2+), TnsA+TnsB alone can execute the DNA breakage and joining reactions of Tn7 recombination. ATP is not essential in this minimal system, revealing that this cofactor is not directly involved in the chemical steps of recombination. In both the TnsAB and TnsABC+D systems, recombination initiates with double-strand breaks at each transposon end that cut Tn7 away from flanking donor DNA. In the minimal system, breakage occurs predominantly at a single transposon end and the subsequent end-joining reactions are intramolecular, with the exposed 3' termini of a broken transposon end joining near the other end of the Tn7 element in the same donor molecule to form circular transposon species. In contrast, in TnsABC+D recombination, breaks occur at both ends of Tn7 and the two ends join to a target site on a different DNA molecule to form an intermolecular simple insertion. This demonstration of the capacity of TnsAB to execute breakage and joining reactions supports the view that these proteins form the Tn7 transposase.

Sleep-disordered breathing in a predominantly African-American pediatric population.

The goal of this study was to characterize sleep and respiratory parameters in children with sleep-disordered breathing (SDB) as compared to children without SDB. Data are from 198 children and adolescents referred for sleep center evaluation, 128 of whom were diagnosed with SDB. In children with SDB, obesity (> 95% wgt for age) was more common than being severely underweight (< 5% wgt for age), but only the older children with SDB were heavier than age-matched normal sleepers. Children with SDB had increased EEG arousals; sleep architecture was not otherwise significantly different from the non-SDB group. African-American children with SDB had significantly greater oxygen desaturation with obstructive events compared to Caucasian and Latino children. It appears that the role of obesity as a risk factor for obstructive sleep apnea (OSA) increases in children above the age of 8-years. Additionally, African-American children with SDB may be at increased risk for hypoxemia and cardiovascular consequences of SDB.

Model of biological evolution with threshold dynamics and infinitely many absorbing states.

We study a model of biological evolution where the survival of a given species depends on its interactions with neighboring species. In the steady state the model has an active phase and an absorbing phase, which are separated by the critical point of the directed percolation universality class. The absorbing phase is infinitely degenerate and the dynamical behavior of our model is found to be nonuniversal.

Chemo- and baroresponses differ in African-Americans and Caucasians in sleep.

To determine sleep effects on baro- and ventilatory responses to transient chemo- and barostimulation in African-Americans and Caucasians, 26 nonobese normotensive young subjects (13 African-Americans and 13 Caucasians) were studied awake and in non-rapid-eye movement (NREM) and rapid-eye-movement sleep during induced transient hypoxemia (N2), hypertension (phenylephrine, PE), and concomitant hypoxemia and hypertension (N2 + PE). Arterial blood pressure was recorded by plethysmographic volume clamp, minute ventilation by pneumotachograph, and arterial O2 saturation by pulse oximeter. For all subjects, chronotropic baroresponse (Deltapulse interval/Deltasystolic blood pressure, where Delta is change) increased with NREM sleep (P = 0.007). Baroresponse slope was greater in Caucasians than in African-Americans (ANOVA, P = 0.02). Hypoxemic ventilatory response (Deltaminute ventilation/Deltaarterial O2 saturation) was greater in African-Americans than in Caucasians in NREM sleep (P = 0.01), as was hypoxemic attenuation of baroresponse (N2 + PE, P = 0.03). These data suggest sleep-related differences in arterial chemo- and baroreceptor responses in normal young African-Americans and Caucasians, which may have implications concerning development of systemic hypertension.

Respiratory and arousal responses to acoustic stimulation.

STUDY OBJECTIVES: Although sleep-related obstructive apnea is most often associated with transient arousal, the impact of this arousal on respiratory control remains unclear. We tested the hypotheses that acoustic arousing stimulation can generate a significant respiratory response during sleep in healthy subjects and that the magnitude or timing of this response is affected by the presence of electrocortical arousal or inhaled carbon dioxide. DESIGN: We employed binaural tone bursts (0.5-s duration, 4-KHz center frequency, 99-s interstimulus interval) to elicit repetitive transient arousals from sleep during nocturnal polysomnographic recordings beginning at 10 PM and ending at 6 AM. PARTICIPANTS: Recordings were conducted in five healthy adult volunteers aged 24 to 37 years. INTERVENTIONS: Inspired gas was alternated between room air and 3% to 7% CO2 (titrated to yield an approximate 50% increase in minute ventilation) at 1-h intervals. MEASUREMENTS AND RESULTS: Each 30-s epoch was scored for sleep/wake stage according to standard criteria. Only results obtained during nonrapid eye movement sleep are presented herein. Tone-evoked arousals were detected by computer analysis as increased EEG frequency occurring within 3 s of acoustic stimulation. For each tone, respiratory parameters for each of three prestimulus and four poststimulus breaths were normalized to the overall mean of prestimulus breaths measured during room air breathing for each subject. Tone bursts elicited repetitive transient arousals with a mean duration of approximately 10 s from all stages of sleep. With respect to the three prestimulus breaths, acoustic stimulation was associated with increased tidal volume and decreased inspiratory duration for at least four breaths. These respiratory responses to acoustic stimulation were not significantly influenced by either presence of transient arousal from sleep or inspired gas. CONCLUSIONS: We conclude that transient EEG arousal may be repeatedly evoked from nonrapid eye movement sleep by transient acoustic stimulation in normal sleepers. This sensory stimulation is associated with augmented ventilation, a response that is not significantly affected by inspired hypercapnia or the presence of generalized EEG arousal.

A lethal mutant of the catabolite gene activator protein CAP of Escherichia coli.

The dimeric catabolite gene activator protein (CAP) of Escherichia coli uses its recognition helix to bind with each subunit the DNA sequence motif 5' G-7T-6G-5A-4 3'. It makes a direct amino acid-base contact with E181 and cytosine in position-5' on the reverse strand. While testing mutants of CAP in position 181 for specificity changes, we found that CAP E181Q is lethal in high amounts for the E. coli strains we used for cloning. We cloned this CAP mutant successfully in cya- strains, where CAP is inactive. Examination of the in vitro binding activities of CAP E181Q, and of in vivo activity when present in low, non-lethal amounts, revealed loss of specificity but not of binding capacity for its DNA targets. It binds well to CAP consensus with G or T in position-5, better to CAP consensus with A, C in position-5, quite well to lambda consensus operator with G in position-7 and rather weakly to lambda consensus.

Respiratory and electrocortical responses to acoustic stimulation.

Although sleep-related obstructive apnea is most often associated with transient arousal, the impact of this arousal on respiratory control remains unclear. We employed binaural tone bursts (.5 second duration) to elicit repetitive transient arousals from sleep during polygraphic recordings in 5 adult volunteers. By this method, we elicited repetitive transient arousals with a mean duration of approximately 10 seconds from all stages of sleep. With respect to the 3 pre-stimulus breaths, acoustic stimulation was associated with increased tidal volume and decreased inspiratory duration for at least 4 breaths. These respiratory responses to acoustic stimulation were not significantly influenced by either presence of transient arousal from sleep or the sleep state from which arousal occurred. We conclude that transient electro-cortical state changes may be repeatedly evoked from all sleep stages by transient acoustic stimulation in normal sleepers. This sensory stimulation represents a significant respiratory stimulus even when generalized arousal from sleep does not occur.

Mutant bZip-DNA complexes with four quasi-identical protein-DNA interfaces.

The complex between the yeast transcriptional activator GCN4 and the palindromic ATF/CREB site 5'- A4T3G2A1C0*G0'T1'C2'A3'T4'-3' shows dyad symmetry. The basic region of GCN4 contains a segment of 18 amino acids with a partially palindromic sequence: N-LKRARNTEA*ARRSRARKL-C. Symmetric residues are underlined. Apart from the ATF/CREB site, GCN4 also binds well to the symmetric variants with guanine in position 4 (5'-G4T3G2A1C0*G0'T1'C2'A3'C4'-3') or thymine in position 0 (5'-A4T3G2A1T0*A0'T1'C2'A3'T4'-3'). The half-sites of these sequences can be regarded as short pseudo-palindromes with central guanine 2/cytosine 2' base pairs. We investigated whether the geometry of the peptide of the basic region of GCN4 could be functionally related to the pseudo-palindromic character of some target half-sites. Since inspection of the X-ray structures of GCN4-DNA complexes reveals that several amino acid-DNA interactions are symmetric within the wild-type half-complexes, we introduced mutations into a GCN4 bZip peptide that improve the symmetry of the peptide. We found that most of the constructs retain specific DNA recognition. For one mutant, we conclude that it is not only capable of forming DNA complexes showing the well-known overall dyad symmetry, but that the protein-DNA interface of each half-complex can be divided further into two quasi-identical, quasi-symmetric substructures.

Palatal muscle electromyogram activity in obstructive sleep apnea.

Eight subjects (5 men, 3 women, ages 27 to 55) with obstructive sleep apnea syndrome (OSAS) were studied to quantify and compare electromyographic (EMG) activity of levator veli palatini (LVP) and palatoglossus (PG), two velopharyngeal muscles, and genioglossus (GG) during obstructive apnea cycles in non-rapid eye movement (NREM) sleep. EMG activity of three successive preapneic breaths, first and last apneic efforts, and three successive postapneic breaths was quantified for each muscle as peak phasic inspiratory EMG normalized as percent activity of the last preapneic breath. In all subjects, apnea onset coincided with simultaneous inspiratory EMG nadir of all three muscles (LVP = 63 +/- 40%, PG = 74 +/- 53%. GG = 83 +/- 48%. mean +/- SD activity of last preapneic breath). Apnea resolution did not occur until inspiratory EMG of all three muscles simultaneously reached maximal activity, at levels significantly greater than preapneic activity as well as activity of the last preapneic effort (LVP = 215 +/- 205%, PG = 227 +/- 240+, GG = 235 +/- 202%, mean +/- SD activity of last preapneic breath, p < 0.05, Fisher's partial least-squares difference [PLSD] test for each muscle). The presence or absence of electroencephalographic arousal at apnea resolution did not influence these patterns of EMG activity. Inspiratory recruitment of velopharyngeal as well as oropharyngeal muscles appears to be associated with upper airway patency during sleep in patients with OSAS.

Effect of induced transient arousal on obstructive apnea duration.

Six untreated male patients (age 19-55 yr) with obstructive sleep apnea underwent nocturnal polysomnography with acoustic stimulation to determine the effect of transient arousal on obstructive apneas during sleep. Binaural tone bursts (25-95 dB) were delivered in late expiration during the second obstructive apnea of a cycle consisting of four consecutive apneas. For the group, stimulated apneas were significantly shorter (P < 0.05, Fisher's protected least significant difference test) than were the unstimulated apneas when transient electrocortical arousal was elicited in both non-rapid-eye-movement (non-REM) sleep [mean 17 +/- 7 (SD) vs. 26 +/- 9, 23 +/- 10, and 26 +/- 12 s for 2nd vs. 1st, 3rd, and 4th apnea, respectively, of each cycle] and REM sleep (mean 19 +/- 10 vs. 35 +/- 15, 45 +/- 18, and 39 +/- 20 s). Without electrocortical arousal, the stimulated apnea was significantly shortened in non-REM (23 +/- 9 vs. 25 +/- 7, 24 +/- 8, and 26 +/- 8 s) but not in REM (32 +/- 16 vs. 37 +/- 12, 32 +/- 15, and 30 +/- 16 s). Tones delivered relatively early and late in the apnea were equally likely to be associated with resolution of the apnea. The nadir of arterial oxygen saturation of hemoglobin was inversely proportional to apnea length, with higher saturation nadirs associated with the stimulated apneas. These data indicate that transient arousal, induced by nonrespiratory stimulation, influences the resolution of obstructive apneas during sleep.

Acoustically induced cortical arousal increases phasic pharyngeal muscle and diaphragmatic EMG in NREM sleep.

Six healthy subjects (3 males, 3 females) were studied to assess phasic inspiratory responses of upper airway (UA) and diaphragm muscles to electrocortical arousal independent of other potential respiratory stimulation. Transient electroencephalographic (EEG) arousal (abrupt EEG frequency shift > or = 3 s without awakening) was induced during supine stage 2 non-rapid-eye-movement (NREM) sleep with binaural tone bursts (0.5 s, 4 kHz, 25-95 dB). Electromyograms (EMG) of levator veli palatini (EMGlvp) and genioglossus (EMGgg) were obtained with intramuscular electrodes, and EMG of diaphragm (EMGdi) was obtained with esophageal electrodes. EMG signals were processed as moving time-averaged inspiratory activity over 100-ms windows. For each arousal, each of five consecutive postarousal breaths (R1-R5) was scored for peak inspiratory phasic EMG and normalized as percent averaged EMG of the three prearousal breaths for all muscles. After arousal, EMGlvp was increased for R1-R5 and EMGgg and EMGdi were increased for R1-R4. The increase in EMGlvp was greater than those of EMGgg and EMGdi for all response breaths. There was a significant increase in EMGlvp in all subjects, and EMGgg and EMGdi were significantly increased in three and two subjects, respectively. These data indicate that isolated transient electrocortical arousal is generally associated with phasic inspiratory recruitment of UA and diaphragm muscles in normal humans during NREM sleep; velopharyngeal muscle recruitment appears to be more consistent and of greater magnitude and duration than that of oropharyngeal muscle or diaphragm. We speculate that transient arousal from sleep may contribute to UA patency independent of chemical and mechanical respiratory stimuli.

Nasal CPAP continues to improve sleep-disordered breathing and daytime oxygenation over long-term follow-up of occlusive sleep apnea syndrome.

To assess the effects of long-term nasal continuous positive airway pressure (CPAP) in occlusive sleep apnea syndrome (OSA), 17 patients with severe symptomatic OSA had repeated spirometry, arterial blood gases, and nocturnal polysomnograms off nasal CPAP after 3 to 46 months of treatment with nasal CPAP. Without loss of weight or change in respiratory mechanics, the ventilatory disturbance index fell from a mean of 87 events per hour to 57 events per hour (p < 0.0001), correlating with an improvement in mean nocturnal desaturation with sleep-disordered breathing events (r = 0.54, p = 0.03). Moreover, the daytime PaO2 rose significantly from a mean of 69 mm Hg to a mean of 82 mm Hg (P = 0.0001) at follow-up. The rise in daytime PaO2 was not only due to the alleviation of daytime hypercapnea observed in eight of nine hypercapneic subjects since the P(A-a)O2 gradient also decreased significantly. The improvement in PaO2 correlated significantly with the number of months of CPAP therapy, suggesting a continuing effect over time (r = 0.58, p = 0.015). These results indicate that there is a reversible element of the severity of OSA and suggest a result of nasal CPAP therapy may be to reverse the adverse and time-dependent effects of hypoxemia and sleep fragmentation on ventilatory control in severe OSA.

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.

Voluntary hyperventilation in obesity hypoventilation.

Arterial blood gas analysis was performed before and after 60 to 90 s of voluntary hyperventilation in 27 consecutive patients with occlusive sleep apnea syndrome (OSA) and daytime hypercapnia. The percentage of fall in PaCO2 from baseline was examined in relationship to age, body mass index, sleep-disordered breathing indices, and pulmonary function variables. In 14 subjects without airflow obstruction, only one individual could not voluntarily hyperventilate into the normal range, whereas 6 of 13 subjects with airflow obstruction could not hyperventilate to eucapnia. The average percentage of fall in PaCO2 was 16 mm Hg (SEM = 1.3 mm Hg). The percentage of fall in PaCO2 correlated significantly with FEV1/FVC ratio (r = 0.47, p = 0.01) and with FEV1 (r = 0.5, p = 0.008). Although the baseline PaCO2 did not correlate with FEV1, the posthyperventilation PaCO2 did (r = 0.54, p = 0.003). Voluntary hyperventilation studies herein suggest a predominant role for impairment of ventilatory control in the maintenance of hypercapnia in OSA since a fall of PaCO2 into the normal range can usually be obtained. The correlation between the percentage of fall in PaCO2 and spirometric measures of respiratory mechanics, as well as the inability of some subjects to normalize the PaCO2 voluntarily suggests an added role for respiratory mechanical impairment in obesity hypoventilation.

Upper airway muscle activity and the thoracic volume dependence of upper airway resistance.

Although a thoracic volume dependence of upper airway resistance and caliber is known to exist in seated subjects, the mechanisms mediating this phenomenon are unknown. To test the hypothesis that actively altered end-expiratory lung volume (EELV) affects upper airway resistance in the supine position and to explore the mechanisms of any EELV-induced resistance changes, we studied five normal males during wakefulness. Supraglottic upper airway resistance (Ruaw) was calculated at an inspiratory flow of 0.1 l/s. The genioglossal electromyogram was obtained with indwelling wire electrodes and processed as moving time average. End-tidal CO2 was monitored by infrared analyzer. Observations were made during four 20-breath voluntary maneuvers: two at high and two at low EELV in each subject. Each maneuver was preceded by a control period at functional residual capacity. At high lung volume the EELV was increased by 2.23 +/- 0.54 (SD) liters; Ruaw decreased to 67.8 +/- 35.1% of control, while tonic and phasic genioglossal activities declined to 79.0 +/- 23.1 and 72.4 +/- 29.8%, respectively. At low lung volume the EELV was decreased by 0.86 +/- 0.23 liters. Ruaw increased to 178.2 +/- 186.8%, while tonic and phasic genioglossal activities increased to 243.0 +/- 139.3 and 249.1 +/- 146.3%, respectively (P less than 0.0001 for all). The findings were not explained by CO2 perturbations or respiratory pattern. Multiple linear regression analysis indicated that the genioglossal responses blunted the EELV-induced changes in upper airway patency.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancers for RNA polymerase I in mouse ribosomal DNA.

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

Successful use of nasal-CPAP for obstructive sleep apnea in Hunter syndrome with diffuse airway involvement.

A patient with Hunter syndrome and diffuse airway obstruction had daytime hypersomnolence, snoring, and alveolar hypoventilation. Polysomnography showed severe obstructive sleep apnea. In the past, all reported cases of sleep apnea in patients with mucopolysaccharidoses had been treated with tonsillectomy/adenoidectomy or tracheostomy. This patient, in whom tracheostomy would have been very difficult due to the diffuse nature of his airway involvement, was successfully treated with high pressure nasal CPAP and supplemental oxygen.

Breath-by-breath interactions between inspiratory and expiratory duration in occlusive sleep apnea.

We examined interactions between inspiratory duration (TI), expiratory duration (TE), and inspiratory (esophageal) pressure (Pes) generation in seven subjects with confirmed occlusive sleep apnea. Breath-by-breath values of TI, TE, and Pes were identified by digital computer during 21 260-s epochs of repetitive occlusive apnea during non-rapid-eye-movement sleep. The control theory of interacting nonlinear oscillators was used to categorize the interaction between TI and TE for each epoch as either 1) synchronization, the strongest possible interaction between biological oscillators; 2) relative entrainment, a moderate interaction between oscillators; or 3) relative coordination, a weak interaction. The latter two interactions were characterized by systemic oscillations in the moving cross-correlation between TI and TE. The relationship between TI and Pes was analyzed in a similar fashion. Significant oscillations were present in all three parameters (P less than 0.0001 for each). We observed significant negative correlations between TI and TE and between TI and Pes (P less than 0.001 for each) when all breaths for all epochs were pooled. In no epoch was there a significant positive correlation between TI and TE or Pes. All three interactions were observed between TI and TE: five epochs of synchronization, nine of relative entrainment, and seven of relative coordination. In contrast, 19 of 21 epochs exhibited synchronization between TI and Pes, with 2 epochs of relative entrainment. The relative frequency of TI vs. Pes synchronization was significantly greater than TI vs. TE synchronization (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)