Circulatory responses to lower body negative pressure in young Afghans and Danes: implications for understanding ethnic effects on blood pressure regulation.

PURPOSE: We have previously shown that Afghans residing in Denmark for at least 12 years exhibit a lower 24-h ambulatory blood pressure compared to Danes. The purpose of this study was to test the hypothesis that the lower blood pressure reflects attenuated compensatory baroreflex responses in the Afghans. METHODS: On a controlled diet (2,822 cal/day, 55-75 mmol + 2 mmol Na+/kg/day), 12 young males of Afghan (Afghans) and 12 young males of Danish (Danes) origin were exposed to a two-step lower body negative pressure (LBNP) protocol of -20 and -50 mmHg, respectively, each of 10-min duration. RESULTS: Afghans had lower 24-h systolic blood pressure compared to Danes (115 ± 2 vs. 123 ± 1 mmHg, p < 0.05). Cardiac output and stroke volume were significantly lower in Afghans compared to Danes prior to and during each level of LBNP. However, it decreased to the same extent in both groups during LBNP. During LBNP of -20 mmHg, plasma noradrenaline concentration and plasma renin activity (PRA) increased significantly only in the Afghans. At LBNP of -50 mmHg plasma noradrenaline concentration and PRA both increased significantly and similarly in the two groups. CONCLUSION: The lower 24-h ambulatory blood pressure in the Afghans is probably caused by a lower stroke volume, which augmented the circulatory and vasoactive hormonal responses to LBNP in the Afghans. The lower stroke volume in Afghans residing in Denmark compared to that of matched native Danes remains to be explained.

Self-assembly of designed coiled coil peptides studied by small-angle X-ray scattering and analytical ultracentrifugation.

α-Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix-containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small-angle X-ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self-assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self-assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC.

Metal ion controlled self-assembly of a chemically reengineered protein drug studied by small-angle X-ray scattering.

Precise control of the oligomeric state of proteins is of central importance for biological function and for the properties of biopharmaceutical drugs. Here, the self-assembly of 2,2'-bipyridine conjugated monomeric insulin analogues, induced through coordination to divalent metal ions, was studied. This protein drug system was designed to form non-native homo-oligomers through selective coordination of two divalent metal ions, Fe(II) and Zn(II), respectively. The insulin type chosen for this study is a variant designed for a reduced tendency toward native dimer formation at physiological concentrations. A small-angle X-ray scattering analysis of the bipyridine-modified insulin system confirmed an organization into a novel well-ordered structure based on insulin trimers, as induced by the addition of Fe(II). In contrast, unmodified monomeric insulin formed larger and more randomly structured assemblies upon addition of Fe(II). The addition of Zn(II), on the other hand, led to the formation of small quantities of insulin hexamers for both the bipyridine-modified and the unmodified monomeric insulin. Interestingly, the location of the bipyridine-modification significantly affects the tendency to hexamer formation as compared to the unmodified insulin. Our study shows how combining a structural study and chemical design can be used to obtain molecular understanding and control of the self-assembly of a protein drug. This knowledge may eventually be employed to develop an optimized in vivo drug release profile.

Accurate stabilities of laccase mutants predicted with a modified FoldX protocol.

Fungal laccases are multicopper enzymes of industrial importance due to their high stability, multifunctionality, and oxidizing power. This paper reports computational protocols that quantify the relative stability (ΔΔG of folding) of mutants of high-redox-potential laccases (TvLIIIb and PM1L) with up to 11 simultaneously mutated sites with good correlation against experimental stability trends. Molecular dynamics simulations of the two laccases show that FoldX is very structure-sensitive, since all mutants and the wild type must share structural configuration to avoid artifacts of local sampling. However, using the average of 50 MD snapshots of the equilibrated trajectories restores correlation (r ~ 0.7-0.9, r(2) ~ 0.49-0.81) and provides a root-mean-square accuracy of ~1.2 kcal/mol for ΔΔG or 3.5 °C for T(50), suggesting that the time-average of the crystal structure is recovered. MD-averaged input also reduces the spread in ΔΔG, suggesting that local FoldX sampling overestimates free energy changes because of neglected protein relaxation. FoldX can be viewed as a simple "linear interaction energy" method using sampling of the wild type and mutant and a parametrized relative free energy function: Thus, we show in this work that a substantial "hysteresis" of ~1 kcal/mol applies to FoldX, and that an improved protocol that reverses calculations and uses the average obtained ΔΔG enhances correlation with the experimental data. As glycosylation is ignored in FoldX, its effect on ΔΔG must be additive to the amino acid mutations. Quantitative structure-property relationships of the FoldX energy components produced a substantially improved laccase stability predictor with errors of ~1 °C for T(50), vs 3-5 °C for a standard FoldX protocol. The developed model provides insight into the physical forces governing the high stability of fungal laccases, most notably the hydrophobic and van der Waals interactions in the folded state, which provide most of the predictive power.

The cAMP response to isoprenaline in mononuclear cells is markedly increased in the presence of platelets.

We have previously shown that the addition of platelets to mononuclear cells (MNC) increases cAMP in MNC. This response may be of interest because the physical interaction between platelets and MNC plays an important role in the inflammatory process. We have now demonstrated that the addition of both isoprenaline and platelets to MNC resulted in a marked amplification of the cAMP response. Prostaglandins, ATP and adenosine and the P-selectin ligand PSGL-1 could not account for the response. No substance was found in the supernatant that could increase cAMP in MNC. W7, a Ca(2+)-calmodulin inhibitor and the addition of EDTA reduced the response to both platelets and isoprenaline. Furthermore, we have demonstrated that mRNA for type I adenylyl cyclase, which is sensitive to Ca(2+), is present in MNC. No increase in Ca(2+) in the cytoplasma in MNC was recorded, however, by quantitative fluorescence microscopy after addition of platelets to MNC. It is possible that there are small increments in Ca(2+) at the binding sites, which we were unable to detect by our technique. Alternatively the binding of platelets to MNC may induce intermolecular interactions in the cell membrane which may facilitate the synthesis of cAMP.

Sympathetic nervous activity decreases during head-down bed rest but not during microgravity.

We tested the hypothesis that sympathoadrenal activity in humans is low during spaceflight and that this effect can be simulated by head-down bed rest (HDBR). Platelet norepinephrine and epinephrine were measured as indexes of long-term changes in sympathoadrenal activity. Ten normal healthy subjects were studied before and during HDBR of 2-wk duration, as well as during an ambulatory study period of a similar length. Platelet norepinephrine concentrations (half-life = 2 days) were studied in five cosmonauts, 2 wk before launch, within 12 h after landing after 11-12 days of flight, and at least 2 wk after return to Earth. Because of the long half-life of platelet norepinephrine, data obtained early after landing would still reflect the microgravity state. Platelet norepinephrine decreased markedly during HDBR (P < 0.001), whereas there were no significant changes when subjects were ambulatory. Platelet epinephrine did not change during HDBR. During microgravity, platelet norepinephrine and epinephrine increased in four of the five cosmonauts. Platelet norepinephrine concentrations expressed in percentage of preflight and pre-HDBR values, respectively, were significantly different during microgravity compared with HDBR [153 +/- 28% (mean +/- SE) vs. 60 +/- 6%, P < 0.004]. Corresponding values for platelet epinephrine were also significant (293 +/- 85 vs. 90 +/- 12%, P < 0.01). The mechanism of the platelet norepinephrine and epinephrine response during spaceflight flight is most likely related to the concomitant decrease in plasma volume. HDBR cannot be applied to simulate changes in sympathoadrenal activity during microgravity.

The human spleen as an erythrocyte reservoir in diving-related interventions.

Twelve subjects without and ten subjects with diving experience performed short diving-related interventions. After labeling of erythrocytes, scintigraphic measurements were continuously performed during these interventions. All interventions elicited a graduated and reproducible splenic contraction, depending on the type, severity, and duration of the interventions. The splenic contraction varied between approximately 10% for "apnea" (breath holding for 30 s) and "cold clothes" (cold and wet clothes applied on the face with no breath holding for 30 s) and approximately 30-40% for "simulated diving" (simulated breath-hold diving for 30 s), "maximal apnea" (breath holding for maximal duration), and "maximal simulated diving" (simulated breath-hold diving for maximal duration). The strongest interventions (simulated diving, maximal apnea, and maximal simulated diving) elicited modest but significant increases in hemoglobin concentration (0.1-0.3 mmol/l) and hematocrit (0.3-1%). By an indirect method, the splenic venous hematocrit was calculated to 79%. No major differences were observed between the two groups. The splenic contraction should, therefore, be included in the diving response on equal terms with bradycardia, decreased peripheral blood flow, and increased blood pressure.

Methyl effect in azumamides provides insight into histone deacetylase inhibition by macrocycles.

Natural, nonribosomal cyclotetrapeptides have traditionally been a rich source of inspiration for design of potent histone deacetylase (HDAC) inhibitors. We recently disclosed the total synthesis and full HDAC profiling of the naturally occurring azumamides ( J. Med. Chem. 2013 , 56 , 6512 ). In this work, we investigate the structural requirements for potent HDAC inhibition by macrocyclic peptides using the azumamides along with a series of unnatural analogues obtained through chemical synthesis. By solving solution NMR structures of selected macrocycles and combining these findings with molecular modeling, we pinpoint crucial enzyme-ligand interactions required for potent inhibition of HDAC3. Docking of additional natural products confirmed these features to be generally important. Combined with the structural conservation across HDACs 1-3, this suggests that while cyclotetrapeptides have provided potent and class-selective HDAC inhibitors, it will be challenging to distinguish between the three major class I deacetylases using these chemotypes.

Stability Mechanisms of Laccase Isoforms using a Modified FoldX Protocol Applicable to Widely Different Proteins.

A recent computational protocol that accurately predicts and rationalizes protein multisite mutant stabilities has been extended to handle widely different isoforms of laccases. We apply the protocol to four isoenzymes of Trametes versicolor laccase (TvL) with variable lengths (498-503 residues) and thermostability (Topt ∼ 45-80 °C) and with 67-77% sequence identity. The extended protocol uses (i) statistical averaging, (ii) a molecular-dynamics-validated "compromise" homology model to minimize bias that causes proteins close in sequence to a structural template to be too stable due to having the benefits of the better sampled template (typically from a crystal structure), (iii) correction for hysteresis that favors the input template to overdestabilize, and (iv) a preparative protocol to provide robust input sequences of equal length. The computed ΔΔG values are in good agreement with the major trends in experimental stabilities; that is, the approach may be applicable for fast estimates of the relative stabilities of proteins with as little as 70% identity, something that is currently extremely challenging. The computed stability changes associated with variations are Gaussian-distributed, in good agreement with experimental distributions of stability effects from mutation. The residues causing the differential stability of the four isoforms are consistent with a range of compiled laccase wild type data, suggesting that we may have identified general drivers of laccase stability. Several sites near Cu, notably 79, 241, and 245, or near substrate, mainly 265, are identified that contribute to stability-function trade-offs, of relevance to the search for new proficient and stable variants of these important industrial enzymes.

Stability mechanisms of a thermophilic laccase probed by molecular dynamics.

Laccases are highly stable, industrially important enzymes capable of oxidizing a large range of substrates. Causes for their stability are, as for other proteins, poorly understood. In this work, multiple-seed molecular dynamics (MD) was applied to a Trametes versicolor laccase in response to variable ionic strengths, temperatures, and glycosylation status. Near-physiological conditions provided excellent agreement with the crystal structure (average RMSD ∼0.92 Å) and residual agreement with experimental B-factors. The persistence of backbone hydrogen bonds was identified as a key descriptor of structural response to environment, whereas solvent-accessibility, radius of gyration, and fluctuations were only locally relevant. Backbone hydrogen bonds decreased systematically with temperature in all simulations (∼9 per 50 K), probing structural changes associated with enthalpy-entropy compensation. Approaching T opt (∼350 K) from 300 K, this change correlated with a beginning "unzipping" of critical ß-sheets. 0 M ionic strength triggered partial denucleation of the C-terminal (known experimentally to be sensitive) at 400 K, suggesting a general salt stabilization effect. In contrast, F(-) (but not Cl(-)) specifically impaired secondary structure by formation of strong hydrogen bonds with backbone NH, providing a mechanism for experimentally observed small anion destabilization, potentially remedied by site-directed mutagenesis at critical intrusion sites. N-glycosylation was found to support structural integrity by increasing persistent backbone hydrogen bonds by ∼4 across simulations, mainly via prevention of F(-) intrusion. Hydrogen-bond loss in distinct loop regions and ends of critical ß-sheets suggest potential strategies for laboratory optimization of these industrially important enzymes.

Blood pressure in Afghan male immigrants to Denmark.

PURPOSE: Immigration from a Third-World society to a Western society can be associated with higher blood pressure and salt sensitivity. We therefore tested whether immigrants from Afghanistan to Denmark compared with non-immigrant Danes exhibit a (i) higher 24-h ambulatory blood pressure (24-h ABP) and (ii) blunted renin response to a change in salt intake. METHODS: Twenty-four-hour ABP was measured in 40 men of Afghan (Afghans) and 40 men of Danish (Danes) origin. Each group was divided into young (20-30 years, n = 20) and middle aged (40-60 years, n = 20). A 3-day low (70 mmol per 24-h) and a 3-day high (250 mmol per 24-h) salt intake were in addition instituted in subgroups of the young groups (n = 18). RESULTS: Young and middle-aged Afghans exhibited a lower 24-h mean arterial pressure (24-h MAP) than the same respective age groups of Danes (83 ± 1 versus 90 ± 1 mm Hg, P<0·05, and 89 ± 2 versus 100 ± 1 mm Hg, P<0·05). 24-h ABP did not change in any of the young groups during increased salt intake, whereas the Danes exhibited a greater decrease in plasma renin activity (PRA) (P<0·05). Plasma noradrenaline (PNA ) was significantly higher among the young Afghans. CONCLUSIONS: Afghan immigrants to Denmark exhibit a lower 24-h ABP than Danes. In young Afghans, PRA is less sensitive to changes in salt intake, while PNA is higher and may reflect their lower systolic blood pressure and/or arterial pulse pressure. Whether these hormonal differences can explain the lower 24-h ABP in Afghans should be further explored.

Cutaneous noradrenaline measured by microdialysis in complex regional pain syndrome during whole-body cooling and heating.

Complex regional pain syndrome (CRPS) is characterised by autonomic, sensory, and motor disturbances. The underlying mechanisms of the autonomic changes in CPRS are unknown. However, it has been postulated that sympathetic inhibition in the acute phase with locally reduced levels of noradrenaline is followed by an up-regulation of alpha-adrenoceptors in chronic CRPS leading to denervation supersensitivity to catecholamines. This exploratory study examined the effect of cutaneous sympathetic activation and inhibition on cutaneous noradrenaline release, vascular reactivity, and pain in CRPS patients and in healthy volunteers. Seven patients and nine controls completed whole-body cooling (sympathetic activation) and heating (sympathetic inhibition) induced by a whole-body thermal suit with simultaneous measurement of the skin temperature, skin blood flow, and release of dermal noradrenaline. CRPS pain and the perceived skin temperature were measured every 5 min during thermal exposure, while noradrenaline was determined from cutaneous microdialysate collected every 20 min throughout the study period. Cooling induced peripheral sympathetic activation in patients and controls with significant increases in dermal noradrenaline, vasoconstriction, and reduction in skin temperature. The main findings were that the noradrenaline response did not differ between patients and controls or between the CRPS hand and the contralateral unaffected hand, suggesting that the evoked noradrenaline release from the cutaneous sympathetic postganglionic fibres is preserved in chronic CRPS patients.

Molecular interactions between barley and oat beta-glucans and phenolic derivatives.

Equilibrium dialysis, molecular modeling, and multivariate data analysis were used to investigate the nature of the molecular interactions between 21 vanillin-inspired phenolic derivatives, 4 bile salts, and 2 commercially available beta-glucan preparations, Glucagel and PromOat, from barley and oats. The two beta-glucan products showed very similar binding properties. It was demonstrated that the two beta-glucan products are able to absorb most phenolic derivatives at a level corresponding to the absorption of bile salts. Glucosides of the phenolic compounds showed poor or no absorption. The four phenolic derivatives that showed strongest retention in the dialysis assay shared the presence of a hydroxyl group in para-position to a CHO group. However, other compounds with the same structural feature but possessing a different set of additional functional groups showed less retention. Principal component analysis (PCA) and partial least-squares regression (PLS) calculations using a multitude of diverse descriptors related to electronic, geometrical, constitutional, hybrid, and topological features of the phenolic compounds showed a marked distinction between aglycon, glucosides, and bile salt retention. These analyses did not offer additional information with respect to the mode of interaction of the individual phenolics with the beta-glucans. When the barley beta-glucan was subjected to enzyme degradation, the ability to bind some but not all of the phenolic derivatives was lost. It is concluded that the binding must be dependent on multiple characteristics that are not captured by a single molecular descriptor.

Change in beta1-adrenergic receptor protein concentration in adipose tissue correlates with diet-induced weight loss.

The aim of the present study was to examine gene expression and protein concentrations of beta(1)- and beta(2)-adrenergic receptors in subcutaneous adipose tissue in obese subjects in response to weight loss. Eighteen obese subjects were studied during diet-induced weight loss. Beta-adrenergic receptor mRNA levels were quantified by reverse transcription-PCR-HPLC. Beta-adrenergic receptor protein concentrations were measured by Western blotting using fluorescence laser scanning for detection. Subjects lost 12.8+/-0.8 kg (mean+/-S.E.M.) during diet treatment. There was a 34% decrease in the beta(1)-adrenergic receptor mRNA level (0.92+/-0.09 compared with 0.61+/-0.06 amol/microg of DNA; P<0.002). Beta(2)-adrenergic receptor mRNA did not decrease significantly. Beta(2)-adrenergic receptor protein concentration decreased 37% (25.5+/-7.1 compared with 16.0+/-5.6 arbitrary units/ng of DNA; P=0.008), whereas beta(1)-adrenergic receptor protein concentration did not decrease significantly. The degree of weight loss was correlated with the concentration of beta(1)-adrenergic receptor protein (r=0.65, P<0.003) and changes in receptor protein concentration (r=0.50, P=0.035) during the very-low-calorie diet. In conclusion, the present study demonstrates a relationship between beta(1)-adrenergic receptor protein concentration in adipose tissue and the degree of weight loss. This relationship is not directly related to energy expenditure and deserves further investigation.

Glycemic control in diabetes in three Danish counties.

BACKGROUND: Hemoglobin A1c (HbA1c) is a proxy measure for glycemic control in diabetes. We investigated the trend for glycemic control in patients from three Danish counties using HbA1c measurements. METHODS: We studied 2454 patients from a population of 807,000 inhabitants for whom routine monitoring of diabetes using HbA1c-DCCT aligned was initiated in 2001. We estimated the incidence of monitored patients in the population. The progress in patients with originally diabetic HbA1c levels was investigated by cumulative probability plots, and the individual trend in clinical outcome was investigated by a modified difference plot. RESULTS: The age-standardized incidence of monitored patients was <0.5% in all regions. Patients with diabetic first HbA1c concentrations (>or=6.62% HbA1c) showed on average 15% improved glycemic control in the first year. Further improvement was limited. The overall percentage above the treatment target (>or=6.62% HbA1c) was 51% in 2003 compared to 59% in 2001, and the percentage with poor glycemic control (>or=10.0% HbA1c) was reduced from 19% to 4%. Of patients with originally diabetic HbA1c levels, 15% showed progress in glycemic control, and 28% reached treatment targets. In patients with originally normal HbA1c, 75% showed an upward trend in HbA1c levels, which reached diabetic concentrations in 17%. CONCLUSION: Patients with diabetic first HbA1c concentrations (>or=6.62% HbA1c) showed on average 15% improved glycemic control in the first year. Further improvement was limited. In individual patients, 75% with originally diabetic HbA1c levels showed improved glycemic control after 3 years, while 78% with originally normal concentrations showed an upward trend in HbA1c levels.