Skin compatibility and efficacy of a cosmetic skin care regimen with licochalcone A and 4-t-butylcyclohexanol in patients with rosacea subtype I.

BACKGROUND: Patients with rosacea often show facial sensitivity to cosmetics or skin care products that can influence the severity of symptoms and exacerbate erythema and inflammation. Nevertheless, special skin care is necessary to address cosmetic concerns and reduce the potential side-effects of topical or oral treatment of the disease. Appropriate skin care should comprise gentle cleansing, effective moisturization, soothing actives, UV protection and concealing pigments to help neutralize the appearance of redness. OBJECTIVE: To determine the compatibility and efficacy of a skin care regimen (consisting of a cleanser, a day care with SPF25 and a night care) containing licochalcone A (Lic A), an anti-irritant from the licorice plant Glycyrrhiza inflata, and 4-t-butylcyclohexanol (SymSitive(®) ), a substance which acts as a sensitivity regulator, in female subjects with clinically determined subtype I rosacea. METHODS: Thirty-two test subjects with mild to moderate rosacea used the skin care regimen daily for 8 weeks. Clinical assessment of erythema, subjective irritation and clinical photography were performed at baseline and after 4 and 8 weeks. Additionally, a quality-of-life questionnaire was filled out by the test subjects at baseline and week 8. The subjects completed a self-assessment questionnaire on product properties after 4 and 8 weeks of product use. RESULTS: Clinical assessments and subject response confirmed very good tolerability of the regimen, a statistically significant improvement in clinical grading for erythema and tactile roughness at weeks 4 and 8 and on telangiectasia at week 8 when compared to baseline scores. A statistically significant improvement in facial redness (a*) values, based on the L*a*b* colorimetric system, was determined at week 4 and 8 in comparison to baseline. No difference in corneometric measurement was detected at week 4 and 8 compared to baseline. CONCLUSION: The skin care regimen was found to be highly compatible with the sensitive facial skin of patients with rosacea subtype I and effective in improving signs of rosacea. Therefore, the daily use of skin care products containing LicA and SymSitive(®) in patients with rosacea improves the overall skin appearance and the quality of life of these patients.

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin.

The gene responsible for Friedreich's ataxia, a disease characterized by neurodegeneration and cardiomyopathy, has recently been cloned and its product designated frataxin. A gene in Saccharomyces cerevisiae was characterized whose predicted protein product has high sequence similarity to the human frataxin protein. The yeast gene (yeast frataxin homolog, YFH1) encodes a mitochondrial protein involved in iron homeostasis and respiratory function. Human frataxin also was shown to be a mitochondrial protein. Characterizing the mechanism by which YFH1 regulates iron homeostasis in yeast may help to define the pathologic process leading to cell damage in Friedreich's ataxia.

Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

Replicative epithelial cell cultures from normal human prostate gland.

Epithelial cell cultures of the normal human prostate gland were established. The subculturing of these cultures was accomplished with a novel nonenzymatic technique. These cultures were defined as normal epithelial cells on the basis of ultrastructure, karyotype, and inability to grow in soft agar.

Chromosomal analysis of human prostatic adenocarcinoma cell lines.

Although detailed cytogenetic analysis has been carried out in many types of cancer, there is little information on the chromosomal makeup of prostatic cancer cells. Karyological analyses of cell lines derived from both metastatic and primary prostatic carcinoma have been carried out by Q-, C-, and sequential banding techniques. The metastatic line, PC-3, isolated from a bone marrow specimen, is an established epithelial line which is tumorigenic in nude, athymic mice and forms colonies in semisolid agar suspension. A subline, PC-3/M, was isolated from a PC-3-induced mouse tumor. Karyotypic analysis of PC-3 by Q- and C-banding showed the cells to be aneuploid at all culture passage levels. The modal chromosome number shifted from 62 to 55 between the 5th and 50th passages. PC-3 has a unique karyotype. Chromosomes 2, 3, 5, 15, and Y were always absent. At least 11 different marker chromosomes were observed. The subline, PC-3/M, had a similar karyotype and retained the parental PC-3 markers. PC-3/M had a more restricted chromosomal frequency distribution range. Nearly 73% of the PC-3/M cells examined had 60 or 61 chromosomes in contrast to the wide distribution seen in PC-3. Silver staining for nucleolus organizer regions indicated that the number of functional nucleolus organizer regions in PC-3 was proportional to the number of acrocentric chromosomes. Banding analysis of PC-5-PI isolated from primary prostatic adenocarcinoma indicated that this line also had a characteristic karyotype with 28% pseudodiploid and 72% pseudotetraploid components. All metaphases examined were partially trisomic in chromosome 9 and lacked a demonstrable Y chromosome.

The effect of mathematics and coordinate system on comparability and "dependencies" of nucleic acid structure parameters.

This paper critically examines the methodologies used to analyze nucleic acid three-dimensional structure based on guidelines set at a 1988 EMBO workshop. The implications of these analyses cannot be fully understood without a thorough knowledge of how the numbers are calculated. This paper addresses one aspect of the calculations, namely the observed correlations between various parameters. These correlations are addressed in the mathematics by explicitly incorporating the concept of a pivot point, which is the point about which a base rotates as it buckles, propeller twists and opens. Pivot points enable one to model the physical motion of bases more accurately. As a result, they greatly reduce and/or eliminate the statistical correlations between rotational and translational parameters found in other approaches. The correlations that are reduced or eliminated are actually artifacts of the mathematics employed and do not reflect true structural properties of nucleic acids. The mathematics we have developed, including the mathematics of pivot points, are presented in the companion paper. Here, we explain how some of the observed correlations occur as a by-product of the method of calculation, while others are truly structural, and we show how optimum pivot points can be determined to minimize artifactual correlations. The observation that experimental bases often rotate about the long axis in a "propeller" motion as well as rotate about the Z-axis of each base, "opening" into the major groove, is evident in the location of the optimum region for the pivot point as determined in this study. We consider locating a pivot point as a calibration step to increase the agreement between physical intuition and the mathematics of our program.

Nucleic acid structure analysis. Mathematics for local Cartesian and helical structure parameters that are truly comparable between structures.

Analyzing nucleic acid structures in a comparable manner has become increasingly important as the number of solved structures has increased. This paper presents the concepts, mathematics, theorems, and proofs that form the basis of a new program to analyze three-dimensional DNA and RNA structures. The approach taken here provides numerical data in accordance with guidelines set at a 1988 EMBO workshop. Mathematical definitions are provided for all local structural parameters described in the guidelines. The definitions satisfy the guideline requirements while preserving the original physical intuition of the parameters. In particular, the rotational parameters are true rotations based on a simple physical model (net rotation at constant angular velocity), not Euler angles or angles between vectors and planes as is the case with other approaches. As a result, the mathematical definitions are symmetrical with the property that a 5 degrees tilt is the same as a 5 degrees roll and a 5 degrees twist, except that the rotations take place about different axes. In other approaches, a 5 degrees tilt can mean a different amount of net rotation than a 5 degrees roll or a 5 degrees twist. A second unique feature of the mathematics is that it explicitly incorporates the concept of a pivot point, which is the point about which a base in a base-pair rotates as it buckles, propeller twists, and opens. Pivot points enable one to model the physical motion of bases more accurately. As a result, they greatly reduce and/or eliminate the statistical correlations between rotational and translational parameters that arise as mathematically induced artifacts in other approaches. This paper, together with the statistical analysis in the companion paper for determining the locations of the pivot points, provides everything needed to understand the output of the program as it relates to individual structures.

A novel antibody-drug conjugate targeting SAIL for the treatment of hematologic malignancies.

Although several new therapeutic approaches have improved outcomes in the treatment of hematologic malignancies, unmet need persists in acute myeloid leukemia (AML), multiple myeloma (MM) and non-Hodgkin's lymphoma. Here we describe the proteomic identification of a novel cancer target, SAIL (Surface Antigen In Leukemia), whose expression is observed in AML, MM, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). While SAIL is widely expressed in CLL, AML, MM, DLBCL and FL patient samples, expression in cancer cell lines is mostly limited to cells of AML origin. We evaluated the antitumor activity of anti-SAIL monoclonal antibodies, 7-1C and 67-7A, conjugated to monomethyl auristatin F. Following internalization, anti-SAIL antibody-drug conjugates (ADCs) exhibited subnanomolar IC50 values against AML cell lines in vitro. In pharmacology studies employing AML cell line xenografts, anti-SAIL ADCs resulted in significant tumor growth inhibition. The restricted expression profile of this target in normal tissues, the high prevalence in different types of hematologic cancers and the observed preclinical activity support the clinical development of SAIL-targeted ADCs.

Transcriptional and translational features of a sporulation gene of Streptomyces griseus.

The nucleotide (nt) sequence of a 2.8-kb fragment of DNA that restores sporulation to one class of bald mutants of Streptomyces griseus revealed an open reading frame (ORF) with the potential to encode a 55.5-kDa polypeptide. The presence of an in-frame TTA in the coding sequence indicated that translation is likely to require the tRNA(Leu)UUA encoded by the bldA gene. Two overlapping transcripts are initiated at transcriptional start points (tsp) separated by 258 nt and are transcribed in the same direction. The downstream tsp lies within the ORF and is followed by a second potential translation initiation site, which would encode a 49.5-kDa polypeptide in the same reading frame as the 55.5-kDa polypeptide. Transcription assays suggested that both tsp functioned during vegetative growth, but the relative abundance of the shorter transcript decreased during the early stages of submerged sporulation. Analysis of sequentially deleted subclones indicated that expression of the longer ORF was necessary to complement bald mutants. The presence of two tsp alternating with potential translation start codons suggests the temporally regulated synthesis of two polypeptides that have identical C termini but different N termini.

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.

Molecular analysis of sporulation in Streptomyces griseus.

Previous evidence suggested that orf1590 from Streptomyces griseus has the potential to encode two polypeptide products from temporally regulated nested open frames (orfs) and that the longer polypeptide may be a DNA-binding protein. We have developed a hypothetical model of the role of orf1590 in sporulation of S. griseus and have begun to test this model by determining the nucleotide sequence of the orf1590 counterpart from Streptomyces coelicolor. The conservation of the helix-turn-helix domain and the two potential translation start codons is consistent with our model. Continued analysis of bald mutants of S. griseus has indicated that several prematurely synthesize sporulation septa and spore walls. One of these nonsporulating strains appears to be a bldA mutant of S. griseus. Complementation analysis suggests that at least three genetic loci are involved in the correct timing of deposition of sporulation septa and wall thickening.

Nucleotide sequence and molecular evolution of two tomato genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase.

We have isolated and sequenced two cDNA clones (LESS5 and LESS17) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase of tomato (Lycopersicon esculentum). At the nucleotide level, the protein-coding regions of these genes are 85% conserved, while the untranslated 3' regions are only 55% conserved. Comparison with rbcS genes from other species of Solanaceae suggests that the tomato LESS5 gene, the Nicotiana tabacum NTSS23 gene and the Petunia hybrida SSU8 gene are orthologous members of the rbcS gene family. In addition, the tomato gene LESS17, and the Petunia hybrida gene SSU611, may also be orthologous, since their untranslated 3' regions are related. There is a large difference between the two tomato rbcS genes in the frequency of the CG dinucleotide. This difference may reflect different levels of methylation, and therefore expression, of the tomato genes. Many of the differences involving the CG dinucleotide can be represented as transitions between C and T on the noncoding strand. Such changes are consistent with observations that methylated cytosines are hot-spots for transitions.

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Long-term facilitation of ventilation in humans during NREM sleep.

The purpose of this study was to determine whether episodic hypoxic exposure would elicit long term facilitation (LTF) of ventilation (V(I)) in sleeping humans. Twenty subjects gave written informed consent. Of these, six subjects were unable to maintain stable stage 2 sleep or deeper for a majority of the experiment and their data were excluded from the analysis. On night 1 after subjects had reached stable sleep (stage 2 or deeper), the subjects breathed room air for 5 minutes, followed by 3 minutes of hypoxia (F(I)O2 = 8%). This sequence was repeated 10 times, and the breathing pattern was observed for a further 60 minutes. Subjects returned to the laboratory for a second visit, which served as a sham night. Instrumentation and study time were the same as on night 1, but subjects breathed room air only. Airflow, tidal volume (V(T)), end tidal O2 and CO2, and estimation of arterial O2 saturation (%) were measured. Seven of the subjects had long-term facilitation (LTF), which was manifested as a significant increase in V(I) that persisted for up to 40 minutes following the last hypoxic exposure. In the other seven subjects, no substantial increase in V(I) was found. We could not explain this difference based on body size (BMI), gender, level of hypoxemia, or magnitude of the hyperpnea during hypoxia. The difference between the two groups was that the LTF group consisted of habitual snorers, and that the NLTF were not inspiratory-flow-limited during the experiment.

Effect of gender on the development of hypocapnic apnea/hypopnea during NREM sleep.

We hypothesized that a decreased susceptibility to the development of hypocapnic central apnea during non-rapid eye movement (NREM) sleep in women compared with men could be an explanation for the gender difference in the sleep apnea/hypopnea syndrome. We studied eight men (age 25-35 yr) and eight women in the midluteal phase of the menstrual cycle (age 21-43 yr); we repeated studies in six women during the midfollicular phase. Hypocapnia was induced via nasal mechanical ventilation for 3 min, with respiratory frequency matched to eupneic frequency. Tidal volume (VT) was increased between 110 and 200% of eupneic control. Cessation of mechanical ventilation resulted in hypocapnic central apnea or hypopnea, depending on the magnitude of hypocapnia. Nadir minute ventilation in the recovery period was plotted against the change in end-tidal PCO(2) (PET(CO(2))) per trial; minute ventilation was given a value of 0 during central apnea. The apneic threshold was defined as the x-intercept of the linear regression line. In women, induction of a central apnea required an increase in VT to 155 +/- 29% (mean +/- SD) and a reduction of PET(CO(2)) by -4.72 +/- 0.57 Torr. In men, induction of a central apnea required an increase in VT to 142 +/- 13% and a reduction of PET(CO(2)) by -3.54 +/- 0.31 Torr (P = 0.002). There was no difference in the apneic threshold between the follicular and the luteal phase in women. Premenopausal women are less susceptible to hypocapnic disfacilitation during NREM sleep than men. This effect was not explained by progesterone. Preservation of ventilatory motor output during hypocapnia may explain the gender difference in sleep apnea.

Oxygen uptake kinetics in cardiac transplant recipients.

Our purpose was to examine the gas exchange response to exercise in heart transplant (HT) patients and to characterize the O2 uptake kinetics (tau VO2) during successive square-wave on-transients from loadless cycling to moderate exercise. We hypothesized that with a slow heart rate response (and O2 transport limitation) O2 kinetics would be slowed but that with a repeated exercise initiated while the heart rate remained elevated the tau VO2 would be faster. Six male HT patients performed two ramp-function tests to determine peak O2 uptake (1.32 +/- 0.23 l/min) and ventilation threshold (1.02 +/- 0.16 l/min). Patients subsequently completed two repeats of a square-wave forcing function and repeated this on 2 days. Alveolar gas exchange was measured breath by breath. A monoexponential fit of signal-averaged data of the first exercise on-transient (between days) yielded a significantly slower tau VO2 in HT subjects than in healthy men (mean age 47 yr; n = 8) (77 +/- 26 vs. 45 +/- 4 s). With successive exercise (2nd transition) initiated while HR remained elevated the tau VO2 of HT patients was 46 +/- 17 s. The faster O2 kinetics of the second transition suggests that O2 delivery was enhanced and therefore that the tau VO2 may reflect bioenergetic processes controlling the rate of oxidative metabolism.

Contribution of diaphragmatic power output to exercise-induced diaphragm fatigue.

In nine normal humans we compared the effects on diaphragm fatigue of whole body exercise to exhaustion (86-93% of maximal O2 uptake for 13.2 +/- 2.0 min) to voluntary increases in the tidal integral of transdiaphragmatic pressure (integral of Pdi) while at rest at the same magnitude and frequency and for the same duration as those during exercise. After the endurance exercise, we found a consistent and significant fall (-26 +/- 2.9%, range -19.2 to -41.0%) in the Pdi response to supramaximal bilateral phrenic nerve stimulation at all stimulation frequencies (1, 10, and 20 Hz). Integral of Pdi.fB (where fB is breathing frequency) achieved during exercise averaged 509 +/- 81.0 cmH2O/min (range 304.0-957.0 cmH2O/min). At rest, voluntary production of integral of Pdi.fB, which was < 550-600 cmH2O/min (approximately 4 times the resting eupenic integral of Pdi.fB or 60-70% of Pdi capacity), did not result in significant diaphragmatic fatigue, whereas sustained voluntary production of integral of Pdi.fB in excess of these threshold values usually did result in significant fatigue. Thus, with few exceptions (5 of 23 tests) the ventilatory requirements of whole body endurance exercise demanded a level of integral of Pdi.fB that, by itself, was not fatiguing. The rested first dorsal interosseous muscle showed no fatigue in response to supramaximal ulnar nerve stimulation after whole body exercise. We postulate that the effects of locomotor muscle activity, such as competition for blood flow distribution and/or extracellular fluid acidosis, in conjunction with a contracting diaphragm account for most of the exercise-induced diaphragm fatigue.

Longitudinal effects of aging on lung function at rest and exercise in healthy active fit elderly adults.

We retested 18 healthy, active, and highly fit [maximal O2 consumption (VO2max) 201 +/- 12% of predicted] older adults over a 6-yr period (mean age 67-->73 yr) to determine the longitudinal effects of aging on lung function at rest and during exercise. In the 6-yr period, total lung capacity (TLC), functional residual capacity, and diffusion capacity did not change; vital capacity, forced expiratory volume in 1 s, and maximal volitional flow rates decreased; and residual volume and closing capacity/TLC increased 11-13%, all of which were greater than predicted from cross-sectional data. At maximum exercise over the 6-yr period, VO2max fell 11.2 +/- 3.4% (45.0-->40.3 ml.kg-1.min-1), six (of 18) subjects showed significant arterial hypoxemia (arterial O2 saturation < or = 92%), and maximum heart rate and minute ventilation-to-O2 consumption ratio (VF/VO2) were unchanged. At any given submaximal work rate, VE and breathing frequency were higher, the degree of expiratory flow limitation increased, and end-expiratory and end-inspiratory lung volumes were unchanged but remained significantly higher relative to young adults. We conclude that in contrast to implications from cross-sectional data, our longitudinal findings demonstrate that habitual physical activity and high aerobic capacity modify neither the normal deterioration in resting lung function nor the increased levels of ventilatory work during exercise that occur with healthy aging over the sixth and seventh decades of life.

Aerobic fitness effects on exercise-induced low-frequency diaphragm fatigue.

We used bilateral phrenic nerve stimulation (BPNS; at 1, 10, and 20 Hz at functional residual capacity) to compare the amount of exercise-induced diaphragm fatigue between two groups of healthy subjects, a high-fit group [maximal O2 consumption (VO2max) = 69.0 +/- 1.8 ml.kg-1.min-1, n = 11] and a fit group (VO2max = 50.4 +/- 1.7 ml.kg-1.min-1, n = 13). Both groups exercised at 88-92% VO2max for about the same duration (15.2 +/- 1.7 and 17.9 +/- 2.6 min for high-fit and fit subjects, respectively, P > 0.05). The supramaximal BPNS test showed a significant reduction (P < 0.01) in the BPNS transdiaphragmatic pressure (Pdi) immediately after exercise of -23.1 +/- 3.1% for the high-fit group and -23.1 +/- 3.8% (P > 0.05) for the fit group. Recovery of the BPNS Pdi took 60 min in both groups. The high-fit group exercised at a higher absolute workload, which resulted in a higher CO2 production (+26%), a greater ventilatory demand (+16%) throughout the exercise, and an increased diaphragm force output (+28%) over the initial 60% of the exercise period. Thereafter, diaphragm force output declined, despite a rising minute ventilation, and it was not different between most of the high-fit and fit subjects. In summary, the high-fit subjects showed diaphragm fatigue as a result of heavy endurance exercise but were also partially protected from excessive fatigue, despite high ventilatory requirements, because their hyperventilatory response to endurance exercise was reduced, their diaphragm was utilized less in providing the total ventilatory response, and possibly their diaphragm aerobic capacity was greater.