The diaphragm and respiratory muscles

A rapid procedure for the purification of the redox-regulated chloroplast fructose-1,6-bisphosphatase [EC 3.1.3.11] from spinach leaf extract to homogeneity is described. No thiol-reducing agents were present during the purification and the enzyme is > 99% in the oxidized form. A rapid procedure to reduce and activate the Fru-1,6-P2ase by dithiothreitol in the absence of thioredoxin is described. Reduction activates the enzyme up to several hundred-fold when assayed at pH 8.0 with 2 mM Mg2+. The activity of the purified oxidized enzyme is unusually sensitive to changes in Mg2+ and H+ concentration. Tenfold changes in Mg2+ or H+ concentration lead to > 100-fold increases in activity. The recoveries of fructose-1,6-bisphosphatase activity as determined by the activity of the oxidized enzyme at pH 8.0/20 mM Mg2+; pH 9.0/2 mM Mg2+; pH 8/2 mM Mg2+ plus 0.1 mM Hg(II) or of the reduced enzyme at pH 8.0/2 mM Mg2+ are similar (approximately 40%) indicating that the major proportion of these activities in a leaf extract is catalyzed by the same enzyme. Moreover, antibodies raised against the purified enzyme inhibit all of the above activities in crude leaf extracts. The kinetic properties of the purified enzyme suggest that the oxidized Mg(2+)-dependent enzyme can play no significant role in photosynthetic carbon assimilation. A survey of some kinetic properties of Fru-1,6-P2ase activity in extracts of various photosynthetic organisms reveals that all 11 species examined possess a redox- and pH/Mg(2+)-stimulated Fru-1,6-P2ase, whereas Fru-1,6-P2ase in extracts of Taxus baccata (a gymnosperm), Chlorella vulgaris (a green alga), and the cyanobacterium Nostoc muscorum were not activated by Hg(II). The heat stability that proved useful in the purification of the spinach enzyme was conserved in both angiosperms and gymnosperms. The oxidized enzyme (which normally has no thiol groups accessible to 5,5'-dithio-bis[2-nitrobenzoic acid]) but not the reduced enzyme can be stimulated many hundred-fold by addition of extraordinarily low concentrations of Hg(II) to a complete assay mixture. With the aid of EDTA as a Hg(II) buffer, half-maximal stimulation was achieved at 2 x 10(-16) M free Hg(II). Methylmercury also stimulates the enzyme many hundred-fold at very low concentrations. The concentration for half-maximal stimulation by methylmercury was determined with a cyanide buffer to be approximately 10(-16) M. This, together with the high affinity of the enzyme for Hg(II), suggests that Hg(II) stimulates the enzyme by binding to an enzyme thiol group that be comes exposed in the catalytically active enzyme, thereby stabilizing the oxidized enzyme in an active conformation. By contrast, in the absence of Fru-1,6-P2 and either Mg2+ or Ca2+, Hg(II) (even at 2 x 10(-16) M) rapidly inactivates the oxidized Fru-1,6-P2ase. This inactivation is similar to the inactivation of Fru-1,6-P2ase that occurred at high pH (> 9) and which is also prevented by Fru-1,6-P2 and either Mg2+ or Ca2+. Although the Hg(II)- and high pH-inactivated oxidized enzyme has no activity, both forms of the enzyme can be activated by reduction. The usefulness of buffers to maintain low, defined Hg(II) and organic mercurial concentrations is discussed.     

Electromyographic activity of respiratory muscles under hyperbaric conditions

Two subjects were exposed to neon gas mixture and two others--to helium one. Electromyographic activity of their breathing muscles differed between the two groups depending on both the gas mixture and the steps of compression. Obvious difficulties of breathing with the neon hyperbaric mixture were accompanied by depressing of the EMG amplitude, episodic breaches or hyperactivation of the EMG. The findings suggest a predominant role of the diaphragm activity in hyperbaric conditions.     

Peripheral and respiratory muscles in chronic heart failure

It is well-established that in patients with congestive heart failure (CHF), exercise is limited by fatigue and shortness of breath. The poor correlation between the fatigue and indices of central haemodynamic function might indicate that peripheral muscle alterations contribute to impaired exercise capacity. Intrinsic abnormalities of the skeletal muscles have been suggested as a possible explanation. Since the shortness of breath correlates poorly with changes in lung function, changes in the respiratory muscles have been investigated. Studies have demonstrated diaphragmatic myopathy and atrophy similar, in part, to the changes in peripheral skeletal muscles. In CHF, type I (slow twitch) fibre atrophy is seen in respiratory as well as in peripheral muscles. The mechanism of these alterations remains to be elucidated. Studies into the mechanism of muscle dysfunction in congestive heart failure are relevant to the prospect of treatment of the changes in peripheral and respiratory muscles.     

Functional magnetic stimulation of the respiratory muscles in dogs

This study assessed the ability of functional magnetic stimulation (FMS) to activate the respiratory muscles in dogs. With the animal supine, FMS of the phrenic nerves using a high-speed magnetic stimulator was performed by placing a round magnetic coil (MC) at the carotid triangle. Following hyperventilation-induced apnea, changes in volume (deltaV) and airway pressure (deltaP) against an occluded airway were determined. FMS of the phrenic nerves produced substantial inspired function (deltaV = 373 +/- 20.5 mL and deltaP = -20 +/- 2.0 cm H2O). After bilateral phrenectomies, maximal inspired deltaV (219 +/- 12.2 mL) and deltaP (-10 +/- 1.0 cm H2O) were produced when the MC was placed near the C6-C7 spinous processes, while maximal expired deltaV (-199 +/- 22.5 mL) and deltaP (11 +/- 2.3 cm H2O) were produced following stimulation near the T9-T10 spinous processes. We conclude: (1) FMS of either the phrenic or upper intercostal nerves results in inspired volume production; (2) FMS of the lower intercostal nerves generates expired volume production; and (3) FMS of the respiratory muscles may be a useful noninvasive tool for artificial ventilation and assisted cough in patients with spinal cord injuries or other neurological disorders.     

Human respiratory muscles: sensations, reflexes and fatiguability

1. Given the importance of the ventilatory 'pump' muscles, it would not be surprising if they were endowed with both sensory and motor specializations. The present review focuses on some unexpected properties of the respiratory muscle system in human subjects. 2. Although changes in blood gas tension were long held not to influence sensation directly, studies in subjects who are completely paralysed show that increases in arterial CO2 levels elicit strong sensations of respiratory discomfort. 3. Stretch reflexes in human limb muscles contain a monosynaptic spinal excitation and a long-latency excitation. However, inspiratory muscles show an initial inhibition when tested with brief airway occlusions during inspiration. This inhibition does not depend critically on input from pulmonary or upper airway receptors. 4. Human inspiratory muscles (including the diaphragm) have been considered to fatigue during inspiratory resistive loading. However, recent studies using phrenic nerve stimulation to test the force produced by the diaphragm show that carbon dioxide retention (hypoventilation) and voluntary cessation of loading occur before the muscles become overtly fatigued.     

In vivo ultrasound assessment of respiratory function of abdominal muscles in normal subjects

Ultrasonography has recently been proposed for assessing changes in thickness and motion of the diaphragm during contraction in humans. Data on ultrasound assessment of abdominal muscles in humans are scarce. We therefore investigated the changes in thickness and the relevant mechanical effects of abdominal muscles using this technique during respiratory manoeuvres in normal subjects. We evaluated the thickness of the abdominal muscle layers in six normal male subjects (aged 26-36 yrs) using a 7.5 MHz B-mode ultrasound transducer. Gastric (Pg) and mouth pressures, muscle thickness of external oblique (EO), internal oblique (IO), transversus abdominis (TA) and rectus abdominis (RA) were assessed at functional residual capacity (FRC), residual volume (RV), total lung capacity (TLC), during progressive (PEEs) and maximal expiratory efforts (MEEs) against a closed airway and during homolateral (HTR) and contralateral (CTR) trunk rotation. Abdominal muscle thickness was found to be reproducible (coefficient of variation and two-way analysis of variance). Compared to FRC, the thickness of IO, TA and RA significantly increased at RV and during MEEs, whereas EO remained unchanged; at TLC, the thickness of IO and TA significantly decreased. During PEEs, a significant relationship between increase in Pg and TA thickness was observed in all subjects, the thickness of the other abdominal muscles being inconsistently related to Pg. Finally, a significant increase in the thickness of IO and EO was found during HTR and CTR, respectively. We conclude that during maximal expiratory manoeuvres, transversus abdominis, internal oblique and rectus abdominis thickened similarly. Transversus abdominis seems to be the major contributor in generating abdominal expiratory pressure during progressive expiratory efforts. External oblique seems to be preferentially involved during trunk rotation. These results suggest the possible value of studying the abdominal muscles by ultrasonography in various respiratory disorders.     

Influence of respiratory afferents upon the proprioceptive reflex of skeletal muscles in healthy humans

Localization and gene expression of pituitary adenylate cyclase activating polypeptide receptor (PACAPR) in the rat retina were studied by immunocytochemistry and in situ hybridization, respectively. Antisera were raised against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR. Strong PACAPR mRNA expression and PACAPR-like immunoreactivity (PACAPR-LI) were detected in ganglion cells, amacrine cells, and in the inner plexiform layer. PACAPR-LI appeared to be concentrated predominantly in the neuronal perikarya and processes. At the ultrastructural level, strong immunostaining for PACAPR was visible in the plasma membranes, rough endoplasmic reticulum and cytoplasmic matrix in neurons. This study provides the basis for a better understanding of the functions of PACAP in the rat retina.     

Complex studies of the action of intercostal muscles and respiratory biomechanics during lung surgery

The Cottle method is the best for correction of anterior nasal septal deformities. Establishment of the lower tunnel however seems unadvisable because of the common damage to the nasopalatine nerve passing through the incisive foramen with occasional permanent sequelae. The procedure also is time-consuming. As a rule spurs or deviations can be dealt with after establishing upper tunnels without perforation of the mucoperichondrial or mucoperiosteal flaps. The method is described in details.     

EMG power spectrum of respiratory and skeletal muscles during static contraction in healthy man

Changes in EMG power spectrum during isometric voluntary contraction maintained until exhaustion in the range of 20-80% MVC were studied in three skeletal muscles (adductor pollicis or AP, vastus lateralis, and medialis) and two respiratory muscles (diaphragm and rectus abdominis). Quantitative EMG analysis consisted of computation of the median frequency (MF) of power spectra and also the continuous measurement of EMG power in two bands of high (EH) and low (EL) frequencies using bandpass filters. This allowed the calculation of the H/L ratio and its time constant of decay rate (TC delta H/L) throughout the sustained static contraction. The main results were: (1) highly significant, positive correlations between TC delta H/L and the maximal MF changes and also the endurance time to fatigue; (2) EMG changes were determined early, within the first 10-20 s of contraction; and (3) EL always increased throughout the fatiguing isometric contraction, but EH changes markedly varied within the five muscle groups studied. These observations are discussed in terms of the differences in muscle fiber composition and also the variations in motor unit recruitment.     

Evaluation of the respiratory inductive plethysmograph in man

We evaluated the respiratory inductive plethysmograph, (RIP), during tidal breathing in eight men and seven women. This device measures movements of the rib cage and abdomen during breathing and adds the signals to produce an indirect record of ventilation. We recorded on magnetic tape, the rib and abdominal signals separately, with a simultaneous ventilation measurement from a pneumotachograph, and analysed them later using a digital computer. One method of calibration, the simultaneous equation technique, was considered in detail. Agreement between the combined RIP signal and a simultaneous ventilation record was normally within 25% except where the calibration was unsatisfactory or the breathing pattern was irregular. The reproducibility of measurements made 5 min apart was good overall, but some subjects showed marked variability. An alternative calibration method which derives indirect isovolume relationships was also examined, but was found to give very erratic results. The most serious error was the alinearity of the rib and abdominal signals, and the problem of describing thoracic movement using only two sensing elements. We conclude that the RIP is a convenient monitor of ventilation during regular tidal breathing, but that it is not quantitative.     

Evaluation of activity-based techniques to identify transient arousal in respiratory sleep disorders

Two fragments, 6.5kb and 4.3kb encoding 5' end and 3' end of delta-endotoxin cryIA(c) gene, respectively, were selected from the Bacillus thuringiensis kurstaki-HD-73 75kb plasmid gene pool using random primer labelling delta-endotoxin cryIA(b) gene probe. The full-length 3.92kb cryIA(c) gene including 5' end 152 bp promoter sequence and 3' end 198 bp terminater sequence was rebuilt after uncoding sequences were deleted. Three kinds of engineering strains harbouring the same recombinant plasmid pHTY1 were obtained after the cryIA(c) gene had been subcloned in shuttle vector pHT3101 and transformed to E. coli NM522, Bacillus subtilis AS1176 and Bacillus thuringiensis crystal-deficient 4D10(H3ab). SDS-PAGE electrophoresis patterns reveal that the cryIA(c) gene expressed the same 133,300 protoxin proteins in all three host systems with the same molecular weight to the crystal protein from the Bacillus thuringiensis kurstaki HD-73. Immuno-assays indicate that the expression proteins can react with antiserum of HD-73 paraspore crystal protein in the same pattern as the natural crystalprotein. Bioassays using crude expressed products from three host strains reveal that they all showed toxicities to second instar larvae of Plutella xylostella, and their LD50 were calculated to be 0.311 micrograms/cm2, 0.02 micrograms/cm2 and 0.017 micrograms/cm2, respectively.     

Evaluation of WHO monoclonal antibody kit for diagnosis of acute respiratory viral infections

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. The cDNA encoding the mature human IVD polypeptide was cloned in a prokaryotic expression vector, but the level of expression in Escherichia coli was extremely low and attempts to purify the enzyme to homogeneity were unsuccessful. To enhance expression, the nucleotide sequence of 22 codons within the 111-bp region at the 5'-end of the cDNA was altered to accommodate E. coli codon usage without altering the amino-acid coding sequence. The altered IVD cDNA was synthesized by PCR, using a primer containing the desired modifications. Following overnight induction of the E. coli transformed with this cDNA, the enzyme was purified to homogeneity using diethylaminoethyl agarose and high-pressure ceramic hydroxyapatite resins. IVD activity was increased 165-fold in the crude extract of cells containing the modified cDNA, as compared to that containing the wild-type cDNA.     

Evaluation of a nasal/oral discriminate sampling system for capnographic respiratory monitoring

Since the early reports of the deleterious health effects of asbestos first appeared in the British literature, the paucity of information on dust levels has been decried. Research into archival material reporting various methods of assessing asbestos dust levels in British factories during the 1930s indicates that these levels were high indeed.     

Control of abdominal muscles

Abdominal muscles serve many roles; in addition to breathing, especially at higher levels of chemical drive or at increased end-expiratory lung volumes, they are responsible for, or contribute to, such protective reflexes as cough, sneeze, and vomiting, generate the high intra-abdominal pressures necessary for defecation and parturition, are active during postural adjustments, and play an essential role in vocalization in many species. Despite this widespread involvement, however, their control has, with rare exceptions, received little attention for two major reasons. First, in most anesthetized or decerebrate preparations, they are relatively inactive at rest, in part because the position of the preparation (supine or prone with abdomen supported), reduces lung volume and, therefore, their activity. Second, unlike phrenic motoneurons innervating the diaphragm, identification of motoneurons to a particular abdominal muscle is difficult. At the lumbar level, a given motoneuron may innervate any one of the four abdominal muscles; at the thoracic level, they are also intermixed with those innervating the intercostals. The two internal muscles, the internal oblique and the transverse abdominis, respond more to increases in chemical or volume-related drive than the two external muscles, the rectus abdominis and external oblique; the basis for this differential sensitivity is unknown. Segmental reflexes at the thoracic and lumbar levels are sufficient to activate abdominal motoneurons in the absence of descending drive but the basis for these reflex effects is also unknown. Neuroanatomical experiments demonstrate many more inputs to, and outputs from, the nucleus retroambigualis, the brainstem region in which the premotor neurons are located, than can be accounted for by their respiratory role alone. These other connections likely subserve activities other than respiration. Studies of the multifunctional roles of the abdominal muscles, on the basis of recent work, hold considerable promise for improving our understanding of their control.     

Evaluation of the respiratory function by lung scintigraphy in patients candidates for pulmonary resection

Sixty-one patients undergoing pulmonary resection were studied pre- and post-operatively by spirometry, arterial gas determination, and quantitative ventilation/perfusion lung scanning. Our results showed that ventilation and/or perfusional scintigraphic scanning is currently the most reliable method in identifying patients at risk for postoperative respiratory insufficiency. Specifically, this technique was successful in detecting pulmonary areas other than those to be resected presenting ventilation or perfusional abnormalities. Therefore, the technique is particularly useful in predicting residual pulmonary function.