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.
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