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Background: A-kinase anchor protein 4 (AKAP4) and its precursor pro-AKAP4 are two major proteins in spermatozoa of rodents and mammals. Although researchers have characterized the AKAP4 expression in various species, the protein's expression in humans has not been described in detail., Objectives: The objective of this study was to characterize human pro-AKAP4 more precisely (notably the definition of its localization and expression levels in human spermatozoa and testes)., Materials and Methods: pro-AKAP4 protein expression levels were assessed by Western blotting. The pro-AKAP4's localization in spermatozoa and testes was determined using immunofluorescence staining and immunogold electron microscopy. Furthermore, pro-AKAP4 protein expression levels were assessed in a series of 77 human semen samples, and associations with semen parameters were evaluated., Results: Western blotting revealed a 100-kDa band in human sperm protein extracts. The pro-AKAP4 was immunolocalized in the fibrous sheath of the flagellum of ejaculated spermatozoa and in elongated spermatids in human testes. A Western blot analysis of 77 normozoospermic semen samples evidenced striking differences in pro-AKAP4 levels from one to another sample (median [interquartile range] integrated optical density = 305 [49-1038]). No correlations were found for pro-AKAP4 levels on one hand and semen volume, sperm concentration, sperm count, sperm motility, or sperm morphology on the other (p > 0.05 for all). However, pro-AKAP4 levels were positively correlated with motility after density gradient centrifugation of the semen (r = 0.224, p = 0.049)., Discussion: AKAP4 protein might be activated as an alternative pathway to rescue sperm motility. In human spermatozoa, pro-AKAP4 might therefore be a 'reservoir' of mature AKAP4., Conclusion: This study generated new knowledge about pro-AKAP4 in human semen, which may be of interest in the management of male infertility., (© 2018 American Society of Andrology and European Academy of Andrology.)

Liver fibrosis can be reversed by removing its causative injuries; however, the molecular mechanisms mediating the resolution of liver fibrogenesis are poorly understood. We investigate the role of a scaffold protein, A-Kinase Anchoring Protein 12 (AKAP12), during liver fibrosis onset, and resolution. Biliary fibrogenesis and fibrosis resolution was induced in wild-type (WT) or AKAP12-deficient C57BL/6 mice through different feeding regimens with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing chow. AKAP12 expression in portal fibroblasts (PFs) and liver sinusoidal endothelial cells (LSECs) gradually decreased as fibrosis progressed but was restored after cessation of the fibrotic challenge. Histological analysis of human liver specimens with varying degrees of fibrosis of different etiologies revealed that AKAP12 expression diminishes in hepatic fibrosis from its early stages onward. AKAP12 KO mice displayed reduced fibrosis resolution in a DDC-induced biliary fibrosis model, which was accompanied by impaired normalization of myofibroblasts and capillarized sinusoids. RNA sequencing of the liver transcriptome revealed that genes related to ECM accumulation and vascular remodeling were mostly elevated in AKAP12 KO samples. Gene ontology (GO) and bioinformatic pathway analyses identified that the differentially expressed genes were significantly enriched in GO categories and pathways, such as the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. Knockdown of the AKAP12 gene in cultured primary PFs revealed that AKAP12 inhibited PF activation in association with the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. Moreover, AKAP12 knockdown in LSECs led to enhanced angiogenesis, endothelin-1 expression and alterations in laminin composition. Collectively, this study demonstrates that AKAP12-mediated regulation of PFs and LSECs has a central role in resolving hepatic fibrosis.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology., (© 2016 Blackwell Verlag GmbH.)

To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objective: The purpose of this study was to investigate the expression of A-kinase anchor protein 95 (AKAP95), cell cycle protein E1 (cyclinE1) and D1 (cyclinD1), and gap junction protein connexin 43 (Cx43) in ovarian cancer tissues, the relationship between four proteins and clinicopathologic parameters, and the correlation between these proteins., Methods: The expression of proteins in 54 cases of ovarian cancer tissues was detected by immunohistochemical method., Results: The positive expression rates of AKAP95, cyclinD1 and cyclinE1 in ovarian cancer tissues were 72.22%, 66.67% and 79.63%, respectively, which were higher than that of ovarian pericarcinoma tissues expressing as 33.33%, 25% and 8.30% (P<0.05). The positive expression rate of Cx43 in ovarian cancer tissues was 40.74%, which was lower than that of ovarian pericarcinoma tissues expressing as 75%; respectively, and the difference was statistically significant between groups (P<0.05). The expression of cyclinD1 in ovarian cancer tissues was related to the histologic type (P<0.05) while it showed no correlation with the degree of differentiation (P>0.05). Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues showed no correlation with the degree of differentiation or the histologic type (P>0.05). Protein expressions of AKAP95, Cx43 and cyclinE1 were correlated with each other (P<0.05), and the expressions of cyclinD1, cyclinE1 and Cx43 were also correlated with each other (P<0.05). However, AKAP95 and cyclinD1 showed no correlation (P>0.05)., Conclusion: AKAP95, cyclinD1 and cyclinE1 play an important role in promoting the process of ovarian cancer formation. The tumor inhibitory effects of Cx43 protein on the pathogenesis of ovarian cancer were weakened. The expression of cyclinD1 in ovarian cancer tissues is related to the histologic type while it shows no correlation with the degree of differentiation. Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues shows no correlation with the degree of differentiation or the histologic type. AKAP95 expression is correlated with Cx43 and cyclinE1 expression; Cx43 expression is correlated with AKAP95, cyclinD1 and cyclinE1 expression; cyclinE1 expression is correlated with AKAP95, Cx43, cyclinD1 expression; cyclinD1 expression is correlated with Cx43 and cyclinE1 expression, while AKAP95 and cyclinD1 show no correlation.

Background: Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs., Methods: A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile., Results: NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs., Conclusion: The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors., (© 2014 The International Society of Dermatology.)

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity., (© 2015 Eur J Oral Sci.)

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: To explore the expression of A-kinase anchor protein 95 (AKAP95), Cyclin D1, Cyclin E1, and Connexin43 (Cx43) in rectal cancer tissues and assess the associations between each of the proteins and pathological parameters, as well as their inter-relationships., Methods: AKAP95, Cyclin D1, Cyclin E1, and Cx43 protein expression rates were evaluated by immunohistochemistry in 50 rectal cancer specimens and 16 pericarcinoma tissues., Results: The positive rates of AKAP95, Cyclin E1, and Cyclin D1 proteins were 54.00 vs. 18.75%, 62.00 vs. 6.25%, and 72.00 vs. 31.25% in rectal cancer specimens and pericarcinoma tissues, respectively, representing statistically significant differences (P < 0.05). The positive rate of Cx43 protein expression in rectal cancer tissues was 44.00% and 62.50% in pericarcinoma tissues, and the difference between them was not significant (P > 0.05). No significant associations were found between protein expression of AKAP95, Cyclin E1, Cyclin D1, and Cx43, and the degree of differentiation, histological type, and lymph node metastasis of rectal cancer (P > 0.05). However, significant correlations were obtained between the expression rates of AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43, respectively (P < 0.05)., Conclusion: AKAP95, Cyclin E1, and Cyclin D1 protein expression rates were significantly higher in rectal cancer tissues compared with pericarcinoma samples, suggesting an association between these proteins and the development and progression of rectal cancer. In addition, the significant correlations between the proteins (AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43) indicate the possible synergistic effects of these factors in the development and progression of rectal cancer.

Glaucoma targets a variety of different tissues located in both anterior (e.g., trabecular meshwork) and posterior (e.g., optic nerve head) ocular segments. The transmission of damage between these structures cannot be simply ascribed to intraocular pressure increase. Recent experimental findings provide evidence for the involvement of molecular mediators including proteins and microRNAs. Aqueous humor protein composition is characteristically altered during glaucoma progression. Immunohistochemistry analyses indicate that proteins characterizing glaucomatous aqueous humor are released by damaged trabecular meshwork. This feature incudes (a) Nestin, involved in stem cell recruitment and glial cell activation; (b) A Kinase anchor protein, released as consequence of mitochondrial damage and Rho activation establishing cell shape and motility; (c) Actin related protein 2/3 complex, involved in actin polymerization and cell shape maintenance. As established both in vitro and in glaucomatous aqueous humor, trabecular meshwork cells damaged by oxidative stress release extracellular microRNAs inducing glial cell activation, an established pathogenic mechanism in neurodegenerative diseases. Released microRNAs include miR-21 (apoptosis), miR-450 (cell aging, maintenance of contractile tone), miR-107 (Nestin expression, apoptosis), miR-149 (endothelia and extracellular matrix homeostasis). Experimental evidences indicate that the uveoscleral pathway, via suprachoroidal space, can provide a potential route of access from the anterior region to the posterior segment of the eye and could represent the path followed by biologic mediators to reach the inner layer of the peripapillary retina and transmit damage signals from the anterior to posterior segment during glaucoma course.

cAMP-dependent protein kinase A (PKA) plays important regulatory roles during mouse spermatogenesis. PKA-mediated signaling has been shown to regulate gene expression, chromatin condensation, capacitation, and motility during sperm development and behavior, although how PKA is regulated in spatiotemporal manners during spermatogenesis is not fully understood. In the present study, we found that PKA subunit isoforms are expressed and localized differently in meiotic and post-meiotic mouse spermatogenic cells. Regulatory subunit I alpha (RIα) is expressed in spermatocytes and round spermatids, where it is localized diffusely throughout the cytoplasm of cells. During late spermiogenesis, RIα abundance gradually decreases. On the other hand, RIIα is expressed constantly throughout meiotic and post-meiotic stages, and is associated with cytoskeletal structures. Among several A kinase anchoring proteins (AKAPs) expressed in the testis, sperm-specific AKAP3 can be found in the cytoplasm of elongating spermatids and interacts with RIα, as demonstrated by both in vivo and in vitro experiments. In mature sperm, AKAP3 is exclusively found in the principal piece of the flagellum, coincident with only RIIα. Mutagenesis experiments further showed that the preferential interactions of AKAP3 with PKA regulatory subunits are mediated by two highly conserved amphipathic peptides located in the N-terminal region of AKAP3. Thus, AKAP3 is a dual-specificity molecule that modulates PKA isotypes in a spatiotemporal manner during mouse spermatogenesis., (© 2014 Wiley Periodicals, Inc.)

Background: Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues., Methods: A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided., Results: We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series., Conclusions: Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.

The amendment of the interpretation of recently published size-exclusion chromatography (SEC) data for A-kinase anchoring protein (AKAP12) on Sephacryl-S400 has led to an increase in the estimated size of the supermolecular state from 840 to at least 6000 kDa. Although size-exclusion chromatography has sufficed to demonstrate unequivocally the existence of this 190-kDa scaffold protein in a supermolecular state, any quantitative estimate of the oligomer stoichiometry is shown to be precluded by failure of this empirical procedure to incorporate allowance for any deviation from globular shape--an important consideration in view of the extended structures exhibited by other extracellular matrix proteins., (Copyright © 2011 John Wiley & Sons, Ltd.)

Non-obstructive azoospermia (NOA) is currently evaluated by the use of conventional histopathological methods. In some cases, focal spermatogenesis is present in the testes of patients with NOA which may be almost undetectable by routine histopathological examinations. Application of molecular markers in semen to predict the spermatogenesis status in the testis will emphasize the probability of finding sperm in NOA testis through further search using TESE or mTESE. Detection of germ cell-specific transcripts in semen is a signal of germ cells present in the testis. In this study, we used molecular methods to evaluate spermatogenesis status in azoospermic men. Semen samples were collected from 203 men with azoospermia. Total RNA was extracted from the semen precipitates. First-strand complementary deoxyribonucleic acid (cDNA) was synthesized by reverse transcriptase then, (RT)-PCRs were carried out using primers for testis stage-specific genes (DAZ, AKAP4, PRM1, and PRM2). Testicular tissue biopsies were used for evaluating spermatogenesis status in testis. Histopathological examination and LH, FSH, and testosterone level measurements (chemiluminescence assay) were performed. The presence of DAZ and PRM2 transcripts in semen significantly indicated the presence of spermatogonia and spermatids in the testicular tissues. Absence of all four markers in semen confirmed the histopathological results corresponding to sertoli cell only syndrome (SCO). Although TESE should not be excluded solely on this criteria, using PRM1, PRM2, AKAP4, and DAZ transcripts in semen would provide a non-invasive molecular diagnostic tool to better counsel patients before undergoing TESE.

Sleep is important for brain function and cognitive performance. Sleep deprivation (SD) may affect subsequent learning capacity and ability to form new memories, particularly in the case of hippocampus-dependent tasks. In the present study we examined whether SD for 6 or 12 h during the normal resting phase prior to learning affects hippocampus-dependent working memory in mice. In addition, we determined effects of SD on hippocampal glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and their regulatory pathways, which are crucially involved in working memory. After 12 h SD, but not yet after 6 h, spatial working memory in a novel arm recognition task was significantly impaired. This deficit was not likely due to stress as corticosterone levels after SD were not significantly different between groups. In parallel with the change in cognitive function, we found that 12 h SD significantly reduced hippocampal AMPA receptor phosphorylation at the GluR1-S845 site, which is important for incorporation of the receptors into the membrane. SD did not affect protein levels of cyclic-AMP-dependent protein kinase A (PKA) or phosphatase calcineurin (CaN), which regulate GluR1 phosphorylation. However, SD did reduce the expression of the scaffolding molecule A-kinase anchoring protein 150 (AKAP150), which binds and partly controls the actions of PKA and CaN. In conclusion, a relatively short SD during the normal resting phase may affect spatial working memory in mice by reducing hippocampal AMPA receptor function through a change in AKAP150 levels. Together, these findings provide further insight into the possible mechanism of SD-induced hippocampal dysfunction and memory impairment.

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development. Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.