Saliva as a medium for aldosterone measurement in repeated sampling studies

BackgroundSaliva is a readily available biological fluid, making it convenient in diagnosis of diseases and in multi-sampling protocols. Several salivary steroids give a useful index of free plasma levels. Increased incidence of primary aldosteronism (PA) in approximately 10% of the hypertensive population has increased interest in the mineralocorticoid aldosterone.MethodsA biotinylated-aldosterone tracer and a commercially available antibody are used in a time-resolved fluorescence immunoassay (TR-FIA) to measure salivary aldosterone (SA). Saliva was collected in various multi-sampling protocols: Investigation of diurnal rhythm in healthy and PA patients, ACTH stimulation test and posture test in healthy subjects.ResultsMethod validation showed a sensitivity of 19 ng/L and intra-/inter-assay precision between 7.2–10.1% and 8.7–15.7%, respectively. SA correlated significantly (y = 0.2995x ± 0.01, r² = 0.60) to plasma aldosterone measured by a commercial radioimmunoassay. SA (median; 95%CI) was at 111 (95–127) ng/L in PA (n = 84) and 50 (44–56) ng/L in healthy subjects (n = 60). After change in posture, aldosterone increased in both, saliva (57 (47–63) ng/L to 95 (84–117) ng/L) and plasma (26 (26–41) ng/L to 135 (110–181) ng/L). Peak levels were reached after 1 h, and were higher in females than in males.ConclusionsSA correlates well to plasma aldosterone and mirrors responses during conditions of stress. SA is significantly higher in PA, and the diurnal rhythm seen in the healthy is blunted in PA. We additionally found gender-dependent differential responses to posture, with higher increases in females. Measurement of aldosterone in saliva presents a useful and convenient method for application in multi-sampling studies.     

Probiotic Features of Lactic Acid Bacteria Isolated from a Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions

Probiotics and Antimicrobial Proteins - The increased consumers’ interest on the positive role of food in wellbeing and health underscores the need to determine new probiotic microorganisms....     

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (∼100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.IDE³ is an ∼110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (∼100 nm) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-β (Aβ), a peptide vital to the progression of Alzheimer disease (7, 8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of Aβ (5, 9–12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter- and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the disulfide bonds of insulin. Structural analysis reveals that human IDE contains a catalytic chamber formed by the internal cavity of two roughly equally sized ∼55-kDa N- and C-terminal halves (IDE-N and IDE-C, respectively) (2). Within this chamber, only one catalytic center exists. However, IDE cleaves insulin at multiple sites on both the insulin A and B chains to completely inactivate this hormone. It remains unclear whether the cleavages of insulin by IDE proceed in a sequential or stochastic manner.IDE represents an emerging protease family that utilizes an enclosed catalytic chamber to selectively recognize and unfold the substrates for their degradation (1). The volume of the enclosed chamber of IDE (∼16,000 ų) allows the preferential exclusion of peptides that are greater than ∼75 amino acids long. This chamber also has unique electrostatic properties; the internal cavity of IDE-N is predominantly negative, whereas that of IDE-C is positive. Inside the catalytic chamber, IDE has an exosite that is an evolutionarily conserved substrate-binding site ∼30 Å away from the catalytic groove. This exosite is used to anchor the N-terminal end of IDE substrates. The unique size, electrostatic potential, and exosite of Ides'' catalytic chamber are postulated as key factors for the selective binding and unfolding of IDE substrates (1, 2, 14). In addition, one common feature among the known IDE substrates is their higher propensity to form amyloid fibers (8). Amyloidogenic peptides tend to unfold by themselves, which could facilitate their unfolding and subsequent cleavage by IDE. However, the molecular basis of how the catalytic chamber of IDE binds, unfolds, and cleaves insulin into pieces and how the flexibility of this substrate contributes to its cleavage by IDE remain elusive.IDE is known to cut insulin at multiple sites, and the resulting cleavage products are quite complex (4, 15–18). Here we took advantage of the high mass accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and the selective targeting of disulfide bonds by electron capture dissociation (ECD) in our mass spectrometry (MS) analysis to unambiguously identify IDE-degraded fragments of human insulin, as well as the time-dependent production of these fragments. We also present a 2.6- Å insulin-bound IDE structure, revealing extensive shape and charge complementarity of the partially unfolded insulin with the enclosed catalytic chamber and a potential path for the unfolding of insulin. Together, our data elucidate the molecular basis by which IDE engulfs, unfolds, and effectively cleaves insulin into pieces.     

Validation of serum IGF-I as a biomarker to monitor the bioactivity of exogenous growth hormone agonists and antagonists in rabbits

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7–3.3% coefficient of variation) and linearity (90.4–105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P<0.01) and were highly correlated (P<0.0001). Treatment with the GHA lowered IGF-I levels from the fourth injection onwards (P<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to treatment with recombinant human GH or a GHA closely mimics the pharmacodynamics seen in humans, suggesting that rabbits are a suitable new model to test human GH agonists and antagonists.KEY WORDS: Pharmacodynamic marker, Acromegaly, Growth hormone deficiency, Animal model     

Structure of substrate-free human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-induced conformational switch of IDE

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-beta (Abeta) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that Abeta is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in Vmax with only minimal changes to Km. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.     

A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: Validation in human and mouse plasma

BackgroundThe small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 μl sample volume.MethodsFollowing solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin–R–phycoerythrin (SA–PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown.ResultsThe assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1–15.6%/9.9–15.8% and testosterone 9.7–10.9%/7.7–11.4%) and correlate significantly to established assays (r = 0.94–0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6× NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261–396) pg/ml to 2008 (875–2467) in male and from 260 (210–576) to 1120 (734–1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4–8.3) ng/ml to 31.8 (30.4–33.9) ng/ml, P < 0.01).ConclusionsWe describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents.     

Quantification of growth hormone in serum by isotope dilution mass spectrometry

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (u_c) of 3.0%, and a limit of quantification (LOQ) of 1.7 μg/L when quantifying T6 as a GH-derived fragment, whereas recovery = 100.7%, SD = 2.4%, u_c = 2.5%, and LOQ = 2.7 μg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.     

Climate drives community‐wide divergence within species over a limited spatial scale: evidence from an oceanic island

Geographic isolation substantially contributes to species endemism on oceanic islands when speciation involves the colonisation of a new island. However, less is understood about the drivers of speciation within islands. What is lacking is a general understanding of the geographic scale of gene flow limitation within islands, and thus the spatial scale and drivers of geographical speciation within insular contexts. Using a community of beetle species, we show that when dispersal ability and climate tolerance are restricted, microclimatic variation over distances of only a few kilometres can maintain strong geographic isolation extending back several millions of years. Further to this, we demonstrate congruent diversification with gene flow across species, mediated by Quaternary climate oscillations that have facilitated a dynamic of isolation and secondary contact. The unprecedented scale of parallel species responses to a common environmental driver for evolutionary change has profound consequences for understanding past and future species responses to climate variation.     

Palaeo‐islands as refugia and sources of genetic diversity within volcanic archipelagos: the case of the widespread endemic Canarina canariensis (Campanulaceae)

Geographical isolation by oceanic barriers and climatic stability has been postulated as some of the main factors driving diversification within volcanic archipelagos. However, few studies have focused on the effect that catastrophic volcanic events have had on patterns of within‐island differentiation in geological time. This study employed data from the chloroplast (cpDNA haplotypes) and the nuclear (AFLPs) genomes to examine the patterns of genetic variation in Canarina canariensis, an iconic plant species associated with the endemic laurel forest of the Canary Islands. We found a strong geographical population structure, with a first divergence around 0.8 Ma that has Tenerife as its central axis and divides Canarian populations into eastern and western clades. Genetic diversity was greatest in the geologically stable ‘palaeo‐islands’ of Anaga, Teno and Roque del Conde; these areas were also inferred as the ancestral location of migrant alleles towards other disturbed areas within Tenerife or the nearby islands using a Bayesian approach to phylogeographical clustering. Oceanic barriers, in contrast, appear to have played a lesser role in structuring genetic variation, with intra‐island levels of genetic diversity larger than those between‐islands. We argue that volcanic eruptions and landslides after the merging of the palaeo‐islands 3.5 Ma played key roles in generating genetic boundaries within Tenerife, with the palaeo‐islands acting as refugia against extinction, and as cradles and sources of genetic diversity to other areas within the archipelago.     

Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme

Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid beta (Abeta). Tight interactions with substrates occur at an exosite located approximately 30 A away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9 A crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite and not to the catalytic site. In agreement with observed high K(m) values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all 13 cysteines is insensitive to the inhibition by S-nitrosoglutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing toward an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.