Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.     

Chromatin structure and dynamics: state-of-the-art

A meeting entitled "Chromatin Structure and Dynamics: State-of-the-Art" organized by Jordanka Zlatanova and Sanford Leuba was held at the NIH from May 8-10, 2002. It was a timely meeting and addressed our current understanding of chromatin structure, dynamics, and function.     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.     

Antibodies in proteomics II: screening,high-throughput characterization and downstream applications

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.     

The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid and efficient endocytosis. Since functionally active CFTR is found in purified clathrin-coated vesicles isolated from both cultured epithelial cells and intact epithelial tissues, we investigated the molecular mechanisms whereby CFTR could enter such endocytic clathrin-coated vesicles. In vivo cross-linking and in vitro pull-down assays show that full-length CFTR binds to the endocytic adaptor complex AP-2. Fusion proteins containing the carboxyl terminus of CFTR (amino acids 1404-1480) were also able to bind AP-2 but did not bind the Golgi-specific adaptor complex AP-1. Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFTR) did not perturb AP-2 binding. Secondary structure predictions suggest that Tyr(1424) is present in a beta-turn conformation, a conformation disrupted by alanine but not phenylalanine. Together, these data suggest that the carboxyl terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP-2 to facilitate efficient entry of CFTR into clathrin-coated vesicles.     

Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.     

Protein kinase-A-mediated secretion of mucin from human colonic epithelial cells

Both Ca(2+)- and cAMP-mediated second messenger cascades acutely regulate mucin secretion from human colonic epithelial cells. To better understand the cAMP-dependent regulation of mucin secretion we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms in mucus-secreting T84 cells, and determined which of these isoforms is responsible for agonist-stimulated mucin secretion. Our results show the presence of both type I and type II PKA in cells that also contain large mucin granules. Forskolin caused a rapid and sustained increase in PKA activity that reached a maximum 5-10 min following its addition. Secretion of mucin was detected 15 min following exposure to forskolin, and continued to increase for a further 15 min before reaching a plateau. Mucin secretion was also measured in the presence of combinations of site-selective cAMP analog pairs, which preferentially activate either type I or type II PKA. Similar levels of mucin secretion were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase mucin secretion. Thus, activation of either type I or type II PKA is able to maximally stimulate secretion of mucins from T84 human colonic epithelial cells.     

The neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.     

Rapid Assessment of Ecosystem Services Provided by Two Mineral Extraction Sites Restored for Nature Conservation in an Agricultural Landscape in Eastern England

Despite growing recognition that mineral sites restored for nature conservation can enhance local biodiversity, the wider societal benefits provided by this type of restoration relative to alternative options are not well understood. This study addresses this research gap by quantifying differences in ecosystem services provision under two common mineral site after-uses: nature conservation and agriculture. Using a combination of site-specific primary field data, benefits transfer and modelling, we show that for our sites restoration for nature conservation provides a more diverse array of ecosystem services than would be delivered under an agricultural restoration scenario. We also explore the effects of addressing different conservation targets, which we find alter the provision of ecosystem services on a service-specific basis. Highly species-focused intervention areas are associated with increased carbon storage and livestock grazing provision, whereas non-intervention areas are important for carbon sequestration, fishing, recreation and flood risk mitigation. The results of this study highlight the wider societal importance of restored mineral sites and may help conservation managers and planners to develop future restoration strategies that provide benefits for both biodiversity and human well-being.