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Humoral factors play an important role in the control of exercise hyperpnea. The role of neuromechanical ventilatory factors, however, is still being investigated. We tested the hypothesis that the afferents of the thoracopulmonary system, and consequently of the neuromechanical ventilatory loop, have an influence on the kinetics of oxygen consumption (VO2), carbon dioxide output (VCO2), and ventilation (VE) during moderate intensity exercise. We did this by comparing the ventilatory time constants (tau) of exercise with and without an inspiratory load. Fourteen healthy, trained men (age 22.6 +/- 3.2 yr) performed a continuous incremental cycle exercise test to determine maximal oxygen uptake (VO2max = 55.2 +/- 5.8 ml x min(-1) x kg(-1)). On another day, after unloaded warm-up they performed randomized constant-load tests at 40% of their VO2max for 8 min, one with and the other without an inspiratory threshold load of 15 cmH2O. Ventilatory variables were obtained breath by breath. Phase 2 ventilatory kinetics (VO2, VCO2, and VE) could be described in all cases by a monoexponential function. The bootstrap method revealed small coefficients of variation for the model parameters, indicating an accurate determination for all parameters. Paired Student's t-tests showed that the addition of the inspiratory resistance significantly increased the tau during phase 2 of VO2 (43.1 +/- 8.6 vs. 60.9 +/- 14.1 s; P < 0.001), VCO2 (60.3 +/- 17.6 vs. 84.5 +/- 18.1 s; P < 0.001) and VE (59.4 +/- 16.1 vs. 85.9 +/- 17.1 s; P < 0.001). The average rise in tau was 41.3% for VO2, 40.1% for VCO2, and 44.6% for VE. The tau changes indicated that neuromechanical ventilatory factors play a role in the ventilatory response to moderate exercise.

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The contribution of respiratory muscle work to the development of the O(2) consumption (Vo(2)) slow component is a point of controversy because it has been shown that the increased ventilation in hypoxia is not associated with a concomitant increase in Vo(2) slow component. The first purpose of this study was thus to test the hypothesis of a direct relationship between respiratory muscle work and Vo(2) slow component by manipulating inspiratory resistance. Because the conditions for a Vo(2) slow component specific to respiratory muscle can be reached during intense exercise, the second purpose was to determine whether respiratory muscles behave like limb muscles during heavy exercise. Ten trained subjects performed two 8-min constant-load heavy cycling exercises with and without a threshold valve in random order. Vo(2) was measured breath by breath by using a fast gas exchange analyzer, and the Vo(2) response was modeled after removal of the cardiodynamic phase by using two monoexponential functions. As anticipated, when total work was slightly increased with loaded inspiratory resistance, slight increases in base Vo(2), the primary phase amplitude, and peak Vo(2) were noted (14.2%, P < 0.01; 3.5%, P > 0.05; and 8.3%, P < 0.01, respectively). The bootstrap method revealed small coefficients of variation for the model parameter, including the slow-component amplitude and delay (15 and 19%, respectively), indicating an accurate determination for this critical parameter. The amplitude of the Vo(2) slow component displayed a 27% increase from 8.1 +/- 3.6 to 10.3 +/- 3.4 ml. min(-1). kg(-1) (P < 0.01) with the addition of inspiratory resistance. Taken together, this increase and the lack of any differences in minute volume and ventilatory parameters between the two experimental conditions suggest the occurrence of a Vo(2) slow component specific to the respiratory muscles in loaded condition.

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To assess the eventual effects of acute oral salbutamol intake on performance and metabolism during submaximal exercise, nine healthy volunteers completed two cycling trials at a power corresponding to 80-85% VO2max, after either placebo (Pla) or salbutamol (Sal, 6 mg) treatment, according to a double-blind randomized protocol. Blood samples were collected both at rest and during exercise (5 min-, 10 min-, 15 min-exhaustion) for C-peptide, FFA, lactate and blood glucose measurements. Cycling performance was significantly improved in the Sal vs. Pla trials (p < 0.05). After Sal intake, resting C-peptide, lactate, FFA and blood glucose values were higher whereas exercise lactate and free fatty acid concentrations were greater during and at the conclusion of the exercise period (p < 0.05). These results suggest that acute salbutamol ingestion improved performance during submaximal exercise probably through an enhancement of the overall contribution to energy production from both aerobic and anaerobic metabolisms.

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BACKGROUND: The seroprevalence of Borrelia burgdorferi infection in risk populations is not well known and varies from area to area due to its marked local trend. OBJECTIVE: To know the seroprevalence of B. burgdorferi infection in persons with high risk for acquiring the disease on account of their outdoor activities, as well as parameters determining that risk. PATIENTS AND METHODS: A cross-sectional study was undertaken with a sample size of 302 voluntary people corresponding to risk populations in the Biscay province by means of an epidemiologic questionnaire as well as serology (ELISA and immunoblot) against B. burgdorferi. RESULTS: A total of 106 individuals (35%) were positive, with no differences regarding sex. The predominant age interval was between 51 and 60 years. The groups with the highest risk for seropositivity were shepherds (42%) and apiculturists (41%). There was a positive correlation with the number of years spent on the activity. Sixty percent of the total studied population remembered a tick bite in the last five years; among seropositive patients, 64% of them remembered that event. There were no differences regarding the type or rural or urban house. However, differences were indeed marked regarding the geographic distribution where the activity took place, which is consistent with the focal distribution of the disease. CONCLUSIONS: Outdoor activities represent a considerable risk factor for acquiring B. burgdorferi infection and a high prevalence (35%) of seropositivity was observed among such groups in the Biscay province. Educational public health campaigns addressed to the different risk groups might help prevent this infection.

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Homeodomains are a class of DNA-binding protein domains which play an important role in genetic regulation in eukaryotes. We have characterized the thermodynamics of folding and sequence-specific association with DNA of the MAT alpha 2 homeodomain of yeast. Using differential scanning and isothermal titration calorimetry, we measured the enthalpy, heat capacity, and Gibbs free energy changes of these processes. The protein-DNA interaction is enthalpically driven at physiological temperatures. DSC data on the process of melting the protein-DNA complex at different salt concentrations were dissected into its endothermic components, yielding the enthalpy change and dissociation constant of binding. A comparison of the circular dichroism spectra of the free and DNA-bound protein species revealed the formation of additional alpha-helical structure upon binding to DNA. We propose that the latter half of helix 3, the recognition helix, is substantially unfolded in the free protein under the conditions used, as has been observed with other homeodomains [Tsao, D. H. H., et al. (1994) Biochemistry 33, 15053-15060: Cox, M., et al. (1995) J. Biomol. NMR 5, 23-32]. Formation of protein structure is induced by DNA binding, and the energies measured for association therefore include a component due to folding.

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We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.

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A valuable approach to understanding the forces that maintain protein structure is to analyze the thermodynamic effects of mutations on protein folding. The folding process is most often described using an energetic model that assumes a two-state transition between the native and denatured states. However, some results obtained using this approach for mutants of the protein staphylococcal nuclease have contradicted expectations from our current understanding of protein energetics. The application of differential scanning calorimetry to a set of mutant nuclease proteins allowed us to measure directly the effects of mutations on the enthalpy and heat capacity changes of unfolding, as well as on the cooperativity. We found that most of these effects can be understood with a three-state model of folding including a distinct intermediate, but not with the two-state model. Use of a three-state instead of a two-state model leads to large differences in conclusions about the stability effects of some mutations, suggesting that reevaluation of the effects of mutations on this and other proteins may be necessary to achieve an accurate description of folding energetics. The two-state assumption commonly used in protein stability studies may be an oversimplification in many cases.

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In a continuation of an earlier study [Carra, J., Anderson, E., & Privalov, P. (1994) Biochemistry 33, 10842-10850], we used differential scanning calorimetry to measure the enthalpy and heat capacity changes of denaturation for 11 mutant forms of staphylococcal nuclease, including the triple mutant [V66L+G88V+G79S]. Several mutant proteins with m- characteristics of guanidinium chloride denaturation were found to denature via a three-state mechanism with increasing temperature. Enthalpy changes for the transitions from the native to intermediate and from the intermediate to denatured states were determined. In the case of the triple mutant, the enthalpy of the second endothermic transition is greater than that of the first. Observation of this second transition provides an explanation for the previously reported large changes in the delta H denaturation for the triple mutant versus wild-type nuclease. The sequence specificity of structure in the intermediate state is discussed with relevance to m values of guanidinium chloride denaturation. The enthalpic level of the intermediate state depends upon the amino acid sequence, suggesting that stabilizing mutations can increase the extent or cohesion of structure present in the intermediate.

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Using microcalorimetry, we found an equilibrium intermediate state during the denaturation of the wild-type and five mutant staphylococcal nuclease proteins: V66L, V66W, G88V, D77A, and E75V. The presence of two distinct heat absorption peaks allowed direct measurement of the enthalpy differences between the native, intermediate, and denatured states. Conditions of low pH and high NaCl concentration facilitated observation of the intermediate, or I-state. We propose to consider the nuclease protein as composed of two subdomains, divided along the active-site cleft. The structure of the I-state apparently consists mainly of the folded beta-barrel subdomain, as does that of a nuclease fragment protein [Shortle, D., & Abeygunawardana, C. (1993) Structure 1, 121-134]. The cooperativity of folding of the subdomains is maintained by electrostatic bonds across the active-site cleft. Removal of these bonds by the mutation D77A or E75V results in decooperation of the protein's structure and a three-state mechanism of denaturation at pH 7.0. The origins of differences in the enthalpy change of denaturation and in the m value of guanidinium chloride-induced denaturation with mutant nucleases are discussed in terms of this three-state mechanism.

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Using high-sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower delta Cp and delta H for the denaturation reaction. The acid-denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid-denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of delta H on pH.

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Staphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded or molten globule form called the "A-state" (Fink et al., 1993, Protein Sci 2:1155-1160). We have found that the A-state of nuclease at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A-state has the far-UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A-state denatures with a sigmoidal change in far-UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A-state in the same way as they affect the denaturation of the native state. The A-state must, therefore, contain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A-state is most stable at temperatures below 0 degrees C.

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With the use of special DNA binding sites, but not the natural aral binding site, the dimeric AraC protein can be forced to make sandwich structures in which two DNA molecules are joined by two AraC protein dimers. Apparently one subunit from each dimer contacts each DNA molecule in an extended structure. These sandwich structures form only in the absence of arabinose. This behavior is consistent with the protein's ability to form DNA loops by binding to separated half sites in the absence of arabinose and its preference for binding to adjacent half-sites in the presence of arabinose.

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The dimeric AraC protein of Escherichia coli binds specifically to DNA sequences upstream of promoters whose transcription is regulated by arabinose. Here we show with affinity measurements, DNase footprinting, dimethyl sulfate premethylation interference and dimethyl sulfate footprinting studies that AraC protein can recognize paired half-sites in direct repeat orientation or inverted repeat orientation. A similar high degree of flexibility was also seen in the ability of the protein in the absence of arabinose to bind tightly and specifically when the separation of its half-sites was increased by 10 or 21 bp. In the presence of arabinose the protein could specifically contact both half-sites of a +10 bp spacing construct but could not contact both in a +21 bp construct. Reduced extensibility of AraC protein in the presence of arabinose provides a simple mechanism for the protein's shift from a non-inducing, DNA looping state to an inducing, non-looping state that contacts two adjacent half-sites at the arapBAD promoter.

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This study compares the effects of two different benzodiazepines used for conscious sedation during combined upper gastrointestinal endoscopy (EGD) and colonoscopy. Subjects were assessed for their degree of analgesia and amnesia for the procedure, prior experience with endoscopy, and willingness to undergo another similar procedure should such be necessary. The patients were randomized single blind to receive either midazolam or diazepam for their preprocedure sedation. The amount of preprocedure sedation utilized was determined by titration of the dose to achieve slurring of speech. Prior to receiving either agent, the subjects were shown a standard card containing pictures of 10 common objects, were asked to name and remember them, and were told they would be "quizzed" (at 30 min and 24 hr) after being sedated for their recollection as to the objects pictured on the card. Each subject filled out a questionnaire addressing their perceived discomfort during the endoscopic procedure and their memory of the procedure 24 hr after the procedure. Sixty-three percent of the midazolam-sedated subjects reported total amnesia for their colonoscopy vs 20% of diazepam-sedated patients (P less than 0.001). Fifty-three percent of midazolam-sedated patients reported total amnesia of their upper gastrointestinal endoscopy vs only 23% of diazepam-sedated subjects (P less than 0.05). The midazolam-sedated subjects reported experiencing less pain with both upper gastrointestinal endoscopy (P less than 0.05) and colonoscopy (P less than 0.001) than did the diazepam-sedated group. Most importantly, the midazolam group was more willing to undergo another similar endoscopic procedure should they be asked to do so by their physician (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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Five liver injury measures (albumin, prothrombin time, gamma globulin, indocyanine green clearance and serum ammonia) were correlated with neuropsychologic test performance in a group of 79 nonalcoholic cirrhotic patients. Employing linear regression analysis techniques revealed that these five measures, particularly albumin and prothrombin time, could explain a significant amount of the variance on neuropsychological tests. The results indicate that functional liver status, even where there is no overt evidence of hepatic encephalopathy, is related to cognitive capacity.

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The neuropsychiatric status of patients with primary biliary cirrhosis (PBC) was contrasted to that of patients with PBC and accompanying Sjogren's syndrome and to a matched group of normal controls. The subjects with PBC + Sjogren's syndrome had a more profound impairment on a battery of psychometric tests than did the subjects with PBC alone. In addition, they more frequently met the criteria for a psychiatric diagnosis of anxiety disorder than did those with PBC alone.

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