Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Quantitative sensory testing measures individual pain responses in emergency department patients

Kevin J Duffy,&nbsp;Katharyn L Flickinger,&nbsp;Jeffrey T Kristan,&nbsp;Melissa J Repine,&nbsp;Alexandro Gianforcaro, Rebecca B Hasley,&nbsp;Saad Feroz,&nbsp;Jessica M Rupp,&nbsp;Jumana Al-Baghli,&nbsp;Maria L Pacella,&nbsp;Brian P Suffoletto,&nbsp;Clifton W Callaway Department of Emergency Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Background: Refining and individualizing treatment of acute pain in the emergency department (ED) is a high priority, given that painful complaints are the most common reasons for ED visits. Few tools exist to objectively measure pain perception in the ED setting. We speculated that variation in perception of fixed painful stimuli would explain individual variation in reported pain and response to treatment among ED patients. Materials and methods: In three studies, we 1) describe performance characteristics of brief quantitative sensory testing (QST) in 50 healthy volunteers, 2) test effects of 10 mg oxycodone versus placebo on QST measures in 18 healthy volunteers, and 3) measure interindividual differences in nociception and treatment responses in 198 ED patients with a painful complaint during ED treatment. QST measures adapted for use in the ED included pressure sensation threshold, pressure pain threshold (PPT), pressure pain response (PPR), and cold pain tolerance (CPT) tests. Results: First, all QST measures had high inter-rater reliability and test&ndash;retest reproducibility. Second, 10 mg oxycodone reduced PPR, increased PPT, and prolonged CPT. Third, baseline PPT and PPR revealed hyperalgesia in 31 (16%) ED subjects relative to healthy volunteers. In 173 (88%) ED subjects who completed repeat testing 30 minutes after pain treatment, PPT increased and PPR decreased (Cohen&rsquo;s dz 0.10&ndash;0.19). Verbal pain scores (0&ndash;10) for the ED complaint decreased by 2.2 (95% confidence intervals [CI]: 1.9, 2.6) (Cohen&rsquo;s dz 0.97) but did not covary with the changes in PPT and PPR (r=0.05&ndash;0.13). Treatment effects were greatest in ED subjects with a history of treatment for anxiety or depression (Cohen&rsquo;s dz 0.26&ndash;0.43) or with baseline hyperalgesia (Cohen&rsquo;s dz 0.40&ndash;0.88). Conclusion: QST reveals individual differences in perception of fixed painful stimuli in ED patients, including hyperalgesia. Subgroups of ED patients with hyperalgesia and psychiatric history report larger treatment effects on ED pain and QST measures. Keywords: emergency department, quantitative sensory testing, hyperalgesia, opioid, anxiety, depressio