In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta-tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

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.

Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses.

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.

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.

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.

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.

Nodular pulmonary sarcoidosis. Clinical, roentgenographic, and physiologic course in five patients.

Five cases of nodular pulmonary sarcoidosis are presented. That nodular infiltration represents a reversible stage of sarcoidosis is suggested by the fact that all patients demonstrated roentgenographic resolution of these infiltrates. In four of the five subjects, tests of pulmonary function showed restrictive disease which remained unchanged or worsened despite radiologic clearing, suggesting persistence of active disease or development of residual pulmonary fibrosis. Evidence of obstruction of airways that was unexplained by a history of smoking was present in all of the five subjects.

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.

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.

Relationship between pulmonary function and sleep-induced respiratory abnormalities.

The relationships between pulmonary function and sleep-induced respiratory events were studied in 34 consecutive male patients with suspected sleep apnea syndrome. In view of the effects of obstructive airway disease on pulmonary volume and airway resistance (Raw), patients were divided into two groups, those with and those without obstructive airway disease. Percent predicted functional residual capacity (FRC) correlated significantly with the apnea-plus-hypopnea index (AHI) in patients with and without obstructive airway disease (r = -0.62 and p less than 0.01; and r = -0.57 and p less than 0.01, respectively) whereas percent predicted total lung capacity had a significant relationship with AHI only in patients with obstructive airway disease (r = -0.80; p less than 0.001). In patients without such disease, a significant correlation between inspiratory airway conductance (Gaw) and AHI (r = -0.47; p less than 0.05) was also present, while percent predicted FRC and Gaw did not correlate with normalized weight. These findings suggest that decreased pulmonary volume and increased Raw contribute to the severity of sleep-induced respiratory abnormalities in patients with sleep apnea syndrome regardless of the degree of obesity.

A comparison of men and women with occlusive sleep apnea syndrome.

Among 118 patients with occlusive sleep apnea syndrome (OSA), defined as daytime hypersomnolence and an apnea hypopnea index (AHI) greater than ten events/h, 41 women were compared with 77 men. Body mass index, spirometric study, PaO2, PaCO2, and results from nocturnal polysomnography were examined in a two-way analysis of variance (ANOVA) for the effects of sex, age group, and a sex-age group interaction. The age groups examined were above and below 42 years, the breakpoint for menopause in the women. Younger persons tended to be more obese and to have a higher AHI. Both sexes had similar pulmonary function, AHI, and nocturnal desaturation, but women experienced significantly fewer completely occluded breathing events and had apneas of shorter mean and maximum duration than men of similar ages. No effect of menopausal status per se was observed. In OSA patients, differences in upper airway occlusion and apnea duration suggest differences between the sexes in upper airway physiology or respiratory control.

Respiratory failure due to Strongyloides stercoralis in a patient with a renal transplant.

We report a case of respiratory failure caused by Strongyloides stercoralis in a patient with a renal transplant; the respiratory failure showed dramatic response to therapy with thiabendazole. The clinical aspects of infestation with S stercoralis in the immunocompromised host are discussed, and features are demonstrated which may have significant implications concerning primary treatment and prophylaxis.

Determinants of hypercapnia in occlusive sleep apnea syndrome.

To assess the relative contributions of age, gender, obesity, pulmonary function, and the severity of sleep-induced respiratory abnormalities to the development of alveolar hypoventilation in patients with occlusive sleep apnea syndrome, prospective data from III patients with occlusive sleep apnea were analyzed by stepwise logistic and multiple regression techniques. The significant variables in a logistic regression model predicting the presence of hypercapnia were daytime arterial oxygen pressure (PaO2; p less than 0.0001) and gender (p less than 0.04), the latter reflecting the higher number of hypercapnic women in our patient population. Multiple regression analysis performed in the hypercapnic group to study the determinants of the severity of elevation of arterial carbon dioxide tension (PaCO2) revealed significant contribution from the PaO2, the apnea-plus-hypopnea index (AHI), and the percent predicted forced vital capacity (r2 = 0.56; p less than 0.0001), whereas in the normocapnic patients, PaCO2 related to PaO2 only. These results suggest that daytime hypoxemia, mechanical impairment of the respiratory system due to obesity or obstructive airway disease (or both), and the severity of sleep-induced respiratory abnormalities as assessed by AHI contribute to the severity of carbon dioxide retention in patients with occlusive sleep apnea in a multifactorial fashion.

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.

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.

Respiratory timing during NREM sleep in patients with occlusive sleep apnea.

To study respiratory timing mechanisms in patients with occlusive apnea, inspiratory and expiratory times (TI and TE) were calculated from the diaphragmatic electromyogram obtained in seven patients during non-rapid-eye-movement (NREM) sleep. Peak diaphragmatic activity (EMGdi) had a curvilinear relationship with TI during the ventilatory and occlusive phases such that TI shortened as EMGdi decreased during the ventilatory phase (r = 0.87, P less than 0.05) and it prolonged as EMGdi increased during the occlusive phase (r = 0.89, P less than 0.02). However, EMGdi vs. TI for the occlusive phase was shifted to the right of that for the ventilatory phase, reflecting the relatively longer TI during upper airway occlusion. TI also had a linear relationship with pleural pressure (r = 0.94, P less than 0.001) that remained unchanged during the ventilatory and occlusive phases such that it prolonged as negative inspiratory pressure increased. These results indicate that respiratory timing is continuously modified in patients with occlusive apnea as inspiratory neural drive fluctuates during NREM sleep and suggest that this modification is due to the net effects of changing inspiratory neural drive and afferent input predominantly from upper airway mechanoreceptors.

Respiratory muscle interaction during NREM sleep in patients with occlusive apnea.

To study respiratory muscle interaction in patients with occlusive apnea, diaphragmatic electromyogram (EMGdi) and gastric, pleural, and transdiaphragmatic pressures (Pga, Ppl, and Pdi, respectively) were studied in seven patients during non-rapid-eye-movement (NREM) sleep. Diaphragmatic force output, as assessed by Pdi, followed the periodic changes in EMGdi but during the occlusive phase the increase in Pdi was more than the increase in EMGdi. This increase in Pdi was essentially due to an increase in Ppl, since Pga and EMGdi had a linear relationship (r = 0.98, P less than 0.001) that did not change during the occlusive and ventilatory phases. Abdominal muscle recruitment evident in Pga and abdominal motion tracings during the occlusive phase when paradoxical rib cage motion was observed suggested that this increase in diaphragmatic efficiency was likely due to a change in diaphragmatic length-tension characteristics. These results demonstrate that, in patients with occlusive apneas, the diaphragm is the predominant respiratory muscle during NREM sleep and that its function is supported by abdominal muscle recruitment.

Respiratory load compensation in chronic airway obstruction.

To assess respiratory neuromuscular function and load compensating ability in patients with chronic airway obstruction (CAO), we studied eight stable patients with irreversible airway obstruction during hyperoxic CO2 rebreathing with and without a 17 cmH2O X l-1 X s flow-resistive inspiratory load (IRL). Minute ventilation (VE), transdiaphragmatic pressure (Pdi), and diaphragmatic electromyogram (EMGdi) were monitored. Pdi and EMGdi were obtained via a single gastroesophageal catheter with EMGdi being quantitated as the average rate of rise of inspiratory (moving average) activity. Based on the effects of IRL on the Pdi response to CO2 [delta Pdi/delta arterial CO2 tension (PaCO2)] and the change in Pdi for a given change in EMGdi (delta Pdi/delta EMGdi) during rebreathing, two groups could be clearly identified. Four patients (group A) were able to increase delta Pdi/delta PaCO2 and delta Pdi/delta EMGdi, whereas in the other four (group B) the IRL responses decreased. All group B patients were hyperinflated having significantly greater functional residual capacity (FRC) and residual volume than group A. In addition the IRL induced percent change in delta Pdi/delta PaCO2, and delta VE/delta PaCO2 was negatively correlated with lung volume so that in the hyperinflated group B the higher the FRC the greater was the decrease in Pdi response due to IRL. In both groups the greater the FRC the greater was the decrease in the ventilatory response to loading. Patients with CAO, even with severe airways obstruction, can effect load compensation by increasing diaphragmatic force output, but the presence of increased lung volume with the associated shortened diaphragm prevents such load compensation.

Dynamics of respiratory drive and pressure during NREM sleep in patients with occlusive apneas.

To study the dynamics of respiratory drive and pressure in patients with occlusive apneas, diaphragmatic electromyogram (EMGdi), esophageal pressure (Pes), and genioglossal electromyogram (EMGge) were monitored during nocturnal sleep in five patients. Both EMGs were analyzed as peak moving time average, and Pes was quantitated as the peak inspiratory change from base line. During the ventilatory phase both EMGs decreased proportionally. The decrease in Pes was less than the decrease observed in EMGdi, and Pes generated for a given EMGdi increased during the preapneic phase in spite of the proportional decrease in EMGdi and EMGge during this period. We conclude that negative inspiratory pressures which lead to the passive collapse of oropharyngeal walls are dependent on both respiratory and upper airway muscle activity and that occlusive apneas of non-rapid-eye-movement (NREM) sleep do occur in spite of proportional changes observed in the activity of both muscle groups. The preapneic increase in negative inspiratory pressures generated for a given respiratory muscle activity is most likely due to the decrease in upper airway muscle activity that is associated with an increase in oropharyngeal resistance.

Induction of periodic breathing during sleep causes upper airway obstruction in humans.

To test the hypothesis that occlusive apneas result from sleep-induced periodic breathing in association with some degree of upper airway compromise, periodic breathing was induced during non-rapid-eye-movement (NREM) sleep by administering hypoxic gas mixtures with and without applied external inspiratory resistance (9 cmH2O X l-1 X s) in five normal male volunteers. In addition to standard polysomnography for sleep staging and respiratory pattern monitoring, esophageal pressure, tidal volume (VT), and airflow were measured via an esophageal catheter and pneumotachograph, respectively, with the latter attached to a tight-fitting face mask, allowing calculation of total pulmonary system resistance (Rp). During stage I/II NREM sleep minimal period breathing was evident in two of the subjects; however, in four subjects during hypoxia and/or relief from hypoxia, with and without added resistance, pronounced periodic breathing developed with waxing and waning of VT, sometimes with apneic phases. Resistive loading without hypoxia did not cause periodicity. At the nadir of periodic changes in VT, Rp was usually at its highest and there was a significant linear relationship between Rp and 1/VT, indicating the development of obstructive hypopneas. In one subject without added resistance and in the same subject and in another during resistive loading, upper airway obstruction at the nadir of the periodic fluctuations in VT was observed. We conclude that periodic breathing resulting in periodic diminution of upper airway muscle activity is associated with increased upper airway resistance that predisposes upper airways to collapse.