Sonographic evaluation of diaphragmatic function during breathing control.

OBJECTIVE: To investigate the effect of "breathing control" on sonographic diaphragmatic excursion. METHOD: A prospective, randomized, assessor-blinded study design involving 20 physiotherapy students; ten with knowledge of the breathing control technique (Group BC) and ten without (Group CON). All participants were asked to perform a Chester step test. Group BC performed BC, while Group CON adopted their own breathing pattern during recovery after the step test. Respiratory rate and sonographic parameters of the diaphragm including diaphragmatic excursion, speed of diaphragmatic contraction (slope of contraction), and inspiratory time were recorded before and after the step test. RESULTS: All baseline data were similar for both groups except age. Respiratory rate at 1 min post-step test was higher in Group CON (24.6±4.9 bpm) compared to Group BC (15.6 ± 3.8 bpm) (p < 0.001). Post-step test sonographic evaluation demonstrated an increase in diaphragmatic excursion with a significant time and group interaction (F(4,72) = 5.499, p = 0.005). Post hoc analysis revealed that the diaphragmatic excursion was significantly higher in Group BC compared to Group CON at first, second and third minute post-step test. Time and group interactions were not significant in inspiration time (F(4,72) = 2.459, p = 0.082) nor the slope of contraction (F(4,72) = 0.655, p = 0.582)]. CONCLUSION: Post-exercise diaphragmatic excursion was higher in participants applying BC. Non-invasive ultrasonography is able to promote objective evaluation of the relationship between breathing techniques and diaphragmatic function.

Verbal numerical scales are as reliable and sensitive as visual analog scales for rating dyspnea in young and older subjects.

This study compared the use of a simple verbal 0-10 numerical rating scale (verbal NRS) and a visual analog scale (VAS) for the rating of dyspnea during exercise in a group of young and older subjects. Twelve younger (32+/-9 yr) and 12 older (71+/-7 yr) subjects used either the verbal NRS or the VAS in a randomised fashion to rate dyspnea during 60 s of uphill treadmill walking (range 5.6-8.8 km h(-1)) performed at either a low (17% grade) or high workload (26% grade) and then during recovery. Rating scales were evaluated twice on separate days (day 1 and day 2) at each workload. While the verbal NRS scores proved to be reliable throughout exercise and recovery, VAS scores were significantly (p<0.05) lower on day 2 during the low workload test (younger group) and the high workload test (older group). Verbal NRS ratings were consistently greater than VAS ratings at both workloads (p<0.001) for both young and older groups. The intra-class correlation coefficients for rating peak dyspnea using either the VAS or verbal NRS were consistently lower for the older subjects (range: r=0.54-0.67) than the younger subjects (range: r=0.70-0.86). Overall, subjects preferred the verbal NRS to the VAS. These results suggest that the verbal NRS compares favourably with the VAS for rating dyspnea during exercise without mask or mouthpiece. However, when rating peak dyspnea both scales appear less reliable when used by the older compared to young subjects.

Ventilatory and gas-exchange responses to incremental exercise performed with reduced muscle glycogen content.

This study examined the relationship between minute ventilation (VE), CO2 production (VCO2), and blood lactate concentration ([La-]) during incremental exercise performed with reduced muscle glycogen stores. Nine untrained female subjects (25.3+/-4.2 year) performed incremental cycling in a normal glycogen (NG) state and under conditions of reduced muscle glycogen (RG) content. To reduce muscle glycogen stores, subjects cycled to exhaustion (124+/-33 min) at a power output corresponding to their gas-exchange anaerobic threshold. Peak oxygen uptake (VO2peak) was unchanged with glycogen reduction, even though subjects achieved a significantly lower maximal power output in the RG state (p<0.05). Peak blood [La-] decreased significantly by 37% in the RG state (p<0.001). At any percentage of VO2peak, O2 uptake and VE were similar for both treatment conditions, whereas VCO2 and respiratory exchange ratio values were lower during the RG trial than under NG conditions. Therefore, VE/VCO2 tended to be higher and end-tidal CO2 partial pressure tended to be lower during exercise performed in the RG state. VE was significantly correlated with VCO2 under both treatment conditions (NG: r=0.99, p<0.01; RG: r=0.99, p<0.01). However, the slope of the VE-VCO2 relationship was significantly elevated during the RG trial (p<0.01). VE during exercise was similar under both treatment conditions, even though VCO2 and blood [La-] were lower during the RG trial compared to the NG trial. This suggests that factors other than CO2 delivery to the lung and metabolic acidosis play an important role in regulating VE during exercise.

Continuous and intermittent exercise responses in individuals with chronic obstructive pulmonary disease.

BACKGROUND: While the acute physiological responses to continuous exercise have been well documented in individuals with chronic obstructive pulmonary disease (COPD), no previous study has examined the response to intermittent exercise in these patients. METHODS: We examined the physiological responses of 10 individuals with moderate COPD (forced expiratory volume in 1 second 52 (15)% predicted) who performed both an intermittent (1 min exercise and rest intervals) and a continuous cycle ergometer test on separate days. Both intermittent and continuous exercise tests were performed at the same power output, calculated as 70% of the peak power attained during an incremental exercise test. RESULTS: Intermittent exercise was associated with significantly lower values for oxygen uptake, carbon dioxide output, expired ventilation, heart rate, plasma lactate concentration, and ratings of breathlessness than continuous exercise. Subjects were able to complete a significantly greater total amount of work during intermittent exercise (71 (32) kJ) than during continuous exercise (31 (24) kJ). The degree of dynamic lung hyperinflation (change in end expiratory lung volume) was significantly lower during intermittent exercise (0.23 (0.07) l) than in continuous exercise (0.52 (0.13) l). CONCLUSIONS: The greater amount of work performed and lower measured physiological responses achieved with intermittent exercise may allow for greater peripheral training adaptations in individuals with more limited lung function. The results suggest that intermittent exercise may be superior to continuous exercise as a mode of training for patients with COPD.

Evolutionarily conserved nuclear migration genes required for early embryonic development in Caenorhabditis elegans.

The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulans nudC3 mutant, demonstrating evolutionary conservation of function. A C. elegans nud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.

The LIS1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to specific subunits of dynein and dynactin and to alpha- and gamma-tubulin.

The NUDF protein of Aspergillus nidulans, which is required for nuclear migration through the fungal mycelium, closely resembles the LIS1 protein required for migration of neurons to the cerebral cortex in humans. Genetic experiments suggested that NUDF influences nuclear migration by affecting cytoplasmic dynein. NUDF interacts with another protein, NUDE, which also affects nuclear migration in A. nidulans. Interactions among LIS1, NUDE, dynein, and gamma-tubulin have been demonstrated in animal cells. In this paper we examine the interactions of the A. nidulans NUDF and NUDE proteins with components of dynein, dynactin, and with alpha- and gamma-tubulin. We show that NUDF binds directly to alpha- and gamma-tubulin and to the first P-loop of the cytoplasmic dynein heavy chain, whereas NUDE binds directly to alpha- and gamma-tubulin, to NUDK (actin-related protein 1), and to the NUDG dynein LC8 light chain. The data suggest a direct role for NUDF in regulation of the dynein heavy chain and an effect on other dynein/dynactin subunits via NUDE. The interactions between NUDE, NUDF, and gamma-tubulin suggest that this protein may also be involved in the regulation of dynein function. Additive interactions between NUDE and dynein and dynactin subunits suggest that NUDE acts as a scaffolding factor between components.

Dyneins have run their course in plant lineage.

Flowering plant genomes lack flagellar and cytoplasmic dyneins as well as the proteins that make up the dynactin complex. The mechanisms for organizing the Golgi apparatus, establishing spindle poles, and moving nuclei, vesicles, and chromosomes in flowering plants must be fundamentally different from those in other systems where these processes are dependent upon dynein and dynactin.

Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA+, related to the cellular DNA damage response.

The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. We are interested in identifying factors, other than topoisomerase I, that are involved in mediating cellular sensitivity to camptothecin. To this end, we have isolated eighteen mutants that are sensitive to camptothecin (sca) in the filamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several different mutagenic agents and to actinomycin D. Using temperature-sensitive mutations in genes that are known to regulate the cell cycle, we showed that the camptothecin sensitivity of the mutant sca299 is not affected by a mitotic block. The abnormal nuclear morphology observed in the sca299 mutant strain suggests that the germlings might be undergoing mitosis in the presence of unrepaired DNA damage, which would result in mitotic catastrophe. The hypersensitivity of the sca299 mutant to camptothecin does not result from elevated levels of topoisomerase I mRNA or from alterations in enzyme activity. Using DNA-mediated complementation of the sca299 mutant phenotype, the scaA+ gene was cloned. This gene encodes a 594-amino acid product; moderate structural similarity suggests that the scaA gene product may be related to the human nibrin gene which encodes a product involved in DNA double-strand break repair. Strains disrupted in the scaA gene were sensitive to the anti-topoisomerase I agent berberine, the DNA crosslinking agents mitomycin C and cis-platinum, and also to t-butyl hydroperoxide, which is an inducer of oxidative stress.

The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics.

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.

Nudf, a fungal homolog of the human LIS1 protein, functions as a dimer in vivo.

The NUDF protein is required for nuclear migration through the mycelium of the filamentous fungus Aspergillus nidulans. It is of particular interest, because it closely resembles a human protein, LIS1, that is required for development of the cerebral cortex. Both are approximately 50-kDa proteins with a short N-terminal predicted coiled coil and seven WD-40 domains in the C-terminal half of the molecule. They also interact with homologous proteins, suggesting that they may have similar biochemical functions. Here we describe the purification to homogeneity of NUDF protein in a single step from a cell-free extract of A. nidulans. We demonstrate that NUDF is a homodimer, that its dimerization occurs via the N-terminal coiled coil region of the molecule, and that it must be a dimer to support the growth of A. nidulans.

The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein.

The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei. NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration. Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly. We screened for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homologue of the nuclear distribution protein RO11 of Neurospora crassa. The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed. A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X. laevis. NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homologue of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil. In addition, NUDF coprecipitates with an epitope-tagged NUDE. The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related.

Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1.

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans, we replaced the gene for the cytoplasmic dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP-nudA. The GFP-NUDA fusion protein is fully functional in vivo: strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP-NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP-NUDA comets after they arrived at the tip was also observed. These dynamics of GFP-NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP-NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP-NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP-NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.

A spindle pole body-associated protein, SNAD, affects septation and conidiation in Aspergillus nidulans.

The nudA1 mutation in the cytoplasmic dynein heavy chain gene inhibits nuclear migration, colony growth and asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans. It also alters the location of the first cell division event (septation) and prevents nucleation of tip cells. We showed previously that a suppressor of nudA1, snaD290, partially reversed the nuclear migration defect and partially restored colony growth. We have now demonstrated that the snaD290 mutation also delays septation and restores the septum to its normal position, allowing tip cells to be nucleated. Although snaD290 does not affect nuclear migration or vegetative hyphal growth, it almost completely inhibits conidiation. We propose that the SNAD protein participates in septation, and is essential for asexual spore formation. SnaD encodes a novel 76-kDa coiled-coil protein (SNAD) that is located at the spindle pole body throughout the cell cycle. Therefore, our results suggest that proteins at the spindle pole body are likely to be involved in temporal regulation of septation in A. nidulans.

Hyphal tip growth and nuclear migration.

Recent molecular and cytological studies have greatly advanced our understanding of hyphal tip growth and nuclear migration in filamentous fungi. Mutants involved in various aspects of hyphal tip growth have been isolated. Genes involved in nuclear migration continue to be identified, including putative regulators. The role of microtubules and microtubule motor proteins in hyphal tip growth has also been studied.

Isolation of a new set of Aspergillus nidulans mutants defective in nuclear migration.

In the filamentous fungus Aspergillus nidulans, nuclear migration in the germ tube is mediated by cytoplasmic dynein. We have previously reported the characterization of four nud (nuclear distribution) genes, nudA, nudC, nudF and nudG, involved in this process. The nudA and nudG genes respectively encode for the heavy chain and the 8-kDa light chain of cytoplasmic dynein. In this work, we describe an improved method for the isolation of nud mutants that has led to the identification of at least ten additional nud loci. We have cloned one of the genes, nudK, and determined that it encodes the actin-related protein Arp1, which is a component of the dynactin complex. This provides the first evidence that dynactin is involved in nuclear migration in A. nidulans.

Mitosis in filamentous fungi: how we got where we are.

This review traces the principal advances in the study of mitosis in filamentous fungi from its beginnings near the end of the 19(th) century to the present day. Meiosis and mitosis had been accurately described and illustrated by the second decade of the present century and were known to closely resemble nuclear divisions in higher eukaryotes. This information was effectively lost in the mid-1950s, and the essential features of mitosis were then rediscovered from about the mid-1960s to the mid-1970s. Interest in the forces that separate chromatids and spindle poles during fungal mitosis followed closely on the heels of detailed descriptions of the mitotic apparatus in vivo and ultrastructurally during this and the following decade. About the same time, fundamental studies of the structure of fungal chromatin and biochemical characterization of fungal tubulin were being carried out. These cytological and biochemical studies set the stage for a surge of renewed interest in fungal mitosis that was issued in by the age of molecular biology. Filamentous fungi have provided model studies of the cytology and genetics of mitosis, including important advances in the study of mitotic forces, microtubule-associated motor proteins, and mitotic regulatory mechanisms.

Nuclear migration, nucleokinesis and lissencephaly.

During the past 20 years, biologists have become used to finding that proteins first identified in simple, genetically manipulable eukaryotic organisms are conserved in higher eukaryotes. This article draws attention to the similarity between NUDF protein, which is required for nuclear migration in the filamentous fungus Aspergillus nidulans, and a mammalian homologue, LIS1, whose malfunction causes lissencephaly, a neuronal migration disease. The authors suggest that there might be an underlying similarity of mechanism between nuclear migration in the fungus and neuronal migration in the brain.

A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.

Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.