Background: Salivary testosterone (Sal-T) may be a useful surrogate of serum free testosterone. The study aims were to use a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to determine whether Sal-T concentrations accurately reflect Sal-T concentrations in both sexes and to investigate practical aspects of sample collection. Methods: Saliva and serum samples were collected in 104 male and 91 female subjects. A more sensitive LC-MS/MS assay was developed to enable Sal-T quantitation in the low concentrations found in females. Saliva (200 mL) was extracted with 1 mL of methyl-tert-butyl ether following the addition of D5-testosterone. Quantitation was performed using a Waters TQ-S mass spectrometer. Results: The assay achieved a lower limit of quantification of 5 pmol/L, sufficiently sensitive to measure testosterone in female saliva. Sal-T showed a diurnal variation but samples taken at weekly and monthly intervals showed no significant differences. Sal-T was stable at ambient temperature for up to 5 days, after freeze-thawing and 3 years frozen storage. Reference intervals for Sal-T were 93-378 pmol/L in males and 5-46 pmol/L in females. Sal-T correlated significantly with serum calculated free-T in males (r ¼ 0.71, P < 0.001) and in females (r ¼ 0.39, P < 0.001). Conclusions: These results confirm that testosterone can be reliably and accurately measured by LC-MS/MS in both adult male and female saliva samples. These results lay the foundation for further exploration of the clinical application of Sal-T as a reliable alternative to serum testosterone in the diagnosis and management of androgen disorders and assessment of androgen status in clinical research.
The overall structure of integrins is that of a ligandbinding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both ␣ and  subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the  subunit. The role played by the knee domains in the regulation of integrinligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the  1 subunit PSI domain and stimulate ligand binding to ␣ 5  1 ; (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the ␣ subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the ␣ subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the ␣ and  subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the ␣ subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.Integrins provide a crucial bridge between the inside and outside environments of the cell by linking the surrounding matrix of a cell to its cytoskeletal framework (1). These receptors are ␣, heterodimers, and both subunits have large extracellular domains and short intracellular regions. Integrins carry a two-way flow of information (inside the cell to out, and outside to in). To achieve this bidirectional signaling integrins must convey shape changes over a long distance, from the intracellular domains to the extracellular regions and vice versa (2, 3). Furthermore, in most cases binding of integrins to their extracellular ligands has to be tightly controlled. For example, the interaction of ␣ IIb  3 with fibrinogen during platelet aggregation needs to be restricted to sites of vessel injury. Regulation of ligand binding is achieved by switching of an integrin between a constitutive low affinity (inactive) state and a high affinity (primed) state. In addition, the interaction of ligands with integrin stabilizes the high affinity state and may cause further shape shifting (ligand-activated state) (4, 5). However, the molecular basis of the conformational changes involved is currently uncertain.The recent crystal structures of the extracellular domains of ␣ V  3 (6, 7) have provided new insights into integrin function. Overall, the integrin structure resembles that of a "head" on two "legs."...
Laminins are large heterotrimeric, multidomain proteins that play a central role in organising and establishing all basement membranes. Despite a total of 45 potential heterotrimeric chain combinations formed through the coiled-coil domain of the 11 identified laminin chains (α1–5, β1–3, γ1–3), to date only 15 different laminin isoforms have been reported. This observation raises the question whether laminin assembly is regulated by differential gene expression or specific chain recognition. To address this issue, we here perform a complete analysis of laminin chain assembly and specificity. Using biochemical and biophysical techniques, all possible heterotrimeric combinations from recombinant C-terminal coiled-coil fragments of all chains were analysed. Apart from laminin 323 (α3, β2, γ3), for which no biochemical evidence of its existence in vivo is available, these experiments confirmed all other known laminin isoforms and identified two novel potential chain combinations, laminins 312 (α3, β1, γ2) and 422 (α4, β2, γ4). Our findings contribute to the understanding of basement membrane structure, function and diversity.
Angiogenesis is essential for tissue repair and regeneration during wound healing but also plays important roles in many pathological processes including tumor growth and metastasis. The receptor protein tyrosine kinase Tie2 and its ligands, the angiopoietins, have important functions in the regulation of angiogenesis. Here, we report a detailed structural and functional characterization of the extracellular region of Tie2. Sequence analysis of the extracellular domain revealed an additional immunoglobulin-like domain resulting in a tandem repeat of immunoglobulin-like domains at the N terminus of the protein. The same domain organization was also found for the Tie1 receptor that shares a high degree of homology with Tie2. Based on structural similarities to other receptor tyrosine kinases and cell adhesion molecules, we demonstrate that the N-terminal two immunoglobulin-like domains of Tie2 harbor the angiopoietin-binding site. Using transmission electron microscopy we furthermore show that the extracellular domain of Tie receptors consists of a globular head domain and a short rod-like stalk that probably forms a spacer between the cell surface and the angiopoietin-binding site. Mutational analysis demonstrated that the head domain consists of the three immunoglobulin-like domains and the three epidermal growth factorlike modules and that the stalk is formed by the three fibronectin type III repeats. These findings might be of particular interest for drug development because Tie receptors are potential targets for treatment of angiogenesis-associated diseases.Proper regulation of angiogenesis is required for the normal growth of embryonic and postnatal tissues as well as physiological processes in the adult such as the continuous remodeling of the female reproductive system and wound healing (1, 2). In contrast, dysregulation of angiogenesis contributes to numerous malignant, ischemic, inflammatory, infectious, and immune disorders including conditions such as diabetic retinopathy and tumor growth and metastasis (1, 2). The angiopoietin (Ang) 3 /Tie signaling system plays important roles in the regulation of angiogenesis (3, 4). Tie1 and Tie2 constitute a family of vascular-specific receptor tyrosine kinases (RTKs) that are expressed mainly on endothelial cells but also in certain hematopoietic cells (5-7). Angiopoietins are a family of four distinct growth factor ligands that bind to Tie2 but not Tie1 (8 -10). They are unique and differ from other growth factors by having opposing effects on receptor phosphorylation. Overexpression and knock-out studies in mice have revealed that Ang1 is an agonist promoting stabilization of vessels by maximizing interactions between endothelial cells and their surrounding support cells and the extracellular matrix (11, 12). In contrast Ang2 was found to be a context-dependent antagonist that promotes angiogenic sprouting or vessel regression depending on the expression of vascular endothelial growth factor-A (13, 14). Ang3 and Ang4 also show context-dependent actions as anta...
Background: Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of testosterone. Variation is seen between assays and this may be due to differences in calibration as commercial calibrators for this assay are not readily available. We investigated the effects calibration in routine clinical LC-MS/MS assays. Methods: All LC-MS/MS users that were registered with the UKNEQAS external quality assurance scheme for testosterone were invited to take part in the study. A set of seven serum samples and serum-based calibrators were sent to all laboratories that expressed an interest. The laboratories were instructed to analyse all samples using there own calibrators and return the results and a method questionnaire for analysis. Results: Fifteen laboratories took part in the study. There was no consensus on supplier of testosterone or matrix for the preparation of calibrators and all were prepared in-house. Also, a wide variety of mass spectrometers, internal standards, chromatography conditions and sample extractions were used. The variation in results did not improve when the results were corrected with a common calibrator. Conclusions: The variation in results obtained could not be attributed to variations in calibrators. The differences in methodologies between laboratories must be the reason for this variation. KeywordsMass spectrometry, laboratory methods, steroid hormones, analytes Accepted: 14th September 2012 Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is now accepted as the preferred technique for the analysis of serum testosterone in both men and particularly women. Despite this, variation can be seen between LC-MS/MS assays on external quality assessment schemes and is most likely to be due to method differences between laboratories.1 One possible area of inconsistency among routine LC-MS/MS assays is the lack of commercially available calibrators. This has previously been shown to have a large effect on LC-MS/MS vitamin D assays. 2,3 We investigated the effects
We have isolated a novel inhibitor of erythropoietic differentiation from the plasma of a patient suffering from idiopathic pure red cell aplasia. This differentiation-inhibiting protein (DIP) specifically blocked the differentiation of human burst-forming unit-erythroid (BFU- E), but not colony-forming unit-erythroid (CFU-E) cells. DIP also blocked the maturation of murine BFU-E cells, but not CFU-E or CFU- granulocyte-macrophage cells, and it inhibited the dimethyl sulfoxide (DMSO)-induced differentiation of Friend murine erythroleukemia cells (FLC) at levels between 10(-10) and 10(-12) mol/L. DIP activity was not detectable in the plasma of normal, healthy subjects. Unlike other known inhibitors of hematopoiesis, DIP appears to directly inhibit erythropoietic differentiation, because it did not affect the proliferation of untreated FLC and it effectively blocked FLC hemoglobinization without affecting the ability of the blocked cells to proliferate. DIP blocked FLC differentiation only when added to the culture medium within 1 hour of inducing the cells with DMSO, suggesting that the protein inhibited an early, but critical, DMSO- induced cellular process. DIP appears to be at least partially responsible for the patient's anemia, and its unique activity suggests a role in the early development of some erythroleukemias.
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