Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow’s udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Introns and reading frames: correlation between splicing sites and their codon positions

Computer analyses of the entire GenBank database were conducted to examine correlation between splicing sites and codon positions in reading frames. Intron insertion patterns (i.e., splicing site locations with respect to codon positions) have been analyzed for all of the 74 codons of all the eukaryote taxonomic groups: primates, rodents mammals, vertebrates, invertebrates, and plants. We found that reading frames are interrupted by an intron at a codon boundary (as opposed to the middle of a codon) significantly more often than expected. This observation is consistent with the exon shuffling hypothesis, because exons that end at codon boundaries can be concatenated without causing a frame shift and thus are evolutionarily advantageous. On the other hand, when introns interrupt at the middles of codons, they exist in between the first and second bases much more frequently than between the second and third bases, despite the fact that boundaries between the first and second bases of codons are generally far more important than those between the second and third bases. The reason for this is not clear and yet to be explained. We also show that the length of an exon is a multiple of 3 more frequently than expected. Furthermore, the total length of two consecutive exons is also more frequently a multiple of 3. All the observations above are consistent with results recently published by Long, Rosenberg, and Gilbert (1995).      

Metabolome and metaboproteome remodeling in nuclear reprogramming

Nuclear reprogramming resets differentiated tissue to generate induced pluripotent stem (iPS) cells. While genomic attributes underlying reacquisition of the embryonic-like state have been delineated, less is known regarding the metabolic dynamics underscoring induction of pluripotency. Metabolomic profiling of fibroblasts vs. iPS cells demonstrated nuclear reprogramming-associated induction of glycolysis, realized through augmented utilization of glucose and accumulation of lactate. Real-time assessment unmasked downregulated mitochondrial reserve capacity and ATP turnover correlating with pluripotent induction. Reduction in oxygen consumption and acceleration of extracellular acidification rates represent high-throughput markers of the transition from oxidative to glycolytic metabolism, characterizing stemness acquisition. The bioenergetic transition was supported by proteome remodeling, whereby 441 proteins were altered between fibroblasts and derived iPS cells. Systems analysis revealed overrepresented canonical pathways and interactome-associated biological processes predicting differential metabolic behavior in response to reprogramming stimuli, including upregulation of glycolysis, purine, arginine, proline, ribonucleoside and ribonucleotide metabolism, and biopolymer and macromolecular catabolism, with concomitant downregulation of oxidative phosphorylation, phosphate metabolism regulation, and precursor biosynthesis processes, prioritizing the impact of energy metabolism within the hierarchy of nuclear reprogramming. Thus, metabolome and metaboproteome remodeling is integral for induction of pluripotency, expanding on the genetic and epigenetic requirements for cell fate manipulation.     

A SINE insertion causes the black-and-tan and saddle tan phenotypes in domestic dogs

Agouti Signaling Protein (ASIP) controls the localized expression of red and black pigment in the domestic dog through interaction with other genes, such as Melanocortin 1 Receptor and Beta-Defensin 103. Specific ASIP alleles are necessary for many of the coat color patterns, such as black-and-tan and saddle tan. Mutations in 2 ASIP alleles, a(y) and a, have previously been identified. Here, we characterize a mutation consisting of a short interspersed nuclear element (SINE) insertion in intron 1 of ASIP that allows for the differentiation of the a(w) wolf sable and a(t) black-and-tan alleles. The SINE insertion is present in dogs with the a(t) and a alleles but absent from dogs with the a(w) and a(y) alleles. Dogs with the saddle tan phenotype were all a(t)/a(t). Schnauzers were all a(w)/a(w). Genotypes of 201 dogs of 35 breeds suggest that there are only 4 ASIP alleles, as opposed to the 5 or 6 predicted in previous literature. These data demonstrate that the dominance hierarchy of ASIP is a(y) > a(w) > a(t) > a.     

Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies

There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The approach would also be very suitable for the analysis of human or experimental antitumor responses.Autoimmune diseases are conditions in which aberrant immune responses cause damage to and dysfunction of the body''s own tissue. They range from prevalent conditions, such as type 1 diabetes mellitus and rheumatoid arthritis, to various types of autoimmune thyroiditis (1), inflammatory bowel diseases (2), skin conditions such as bullous pemphigoid (3), and rarer neurological disorders such as myasthenia gravis (4).Understanding of most of these diseases is still highly incomplete. Fundamental knowledge includes the identity of the antigenic target of the immune response and whether the response is predominantly T cell- or antibody-mediated. In some of the above examples, “candidate” antigens have been proposed as a result of study of the pathophysiology of the disease (e.g. see Ref. 5). The detection of a disease-specific autoantibody allows the development of diagnostic tests, and if the target is a cell surface protein it usually implies that the disease will respond clinically to treatments that reduce the levels of the pathogenic antibodies.In recent years, there has also been increasing interest in natural (or experimental) immune responses to tumor cells that may slow the growth or spread of a tumor. In some cases, however, this immune response may result in pathogenic autoimmunity. For example, antibodies directed to voltage-gated calcium channels expressed on the surface of small cell lung cancer cells can cause neurological dysfunction by binding to similar calcium channels on the motor nerve endings (see Ref. 4). In other cancer-associated (paraneoplastic) disorders, however, there are antibodies to intracellular antigens, which are also shared between the tumor and neuronal tissue, that are highly useful as diagnostic markers for the disorders. In these patients, T cell immunity is thought to be responsible for the neurological disease (see Ref. 6), which generally does not improve with immunosuppressive treatments.Attempts to identify autoantigens and tumor antigens in many autoimmune and cancer-related syndromes have generally used techniques involving screening of mRNA expression libraries or, more recently, separation of soluble extracts of tissue or cell lines by one- or two-dimensional electrophoresis and blotting of the separated proteins onto membranes where they are probed with patient sera. Typically in any one experiment, a large number of protein bands or spots are bound by serum antibodies, and some of the corresponding bands or spots on the gel are then excised, digested, and analyzed by mass spectrometry (e.g. Refs. 7 and 8). The identified proteins have been claimed as novel antigens associated with the condition with sometimes a whole array of proteins identified from a single experiment and claimed to represent a disease-associated “autoimmune profile.” However, the identified proteins are often common intracellular proteins with the same or closely related proteins repeatedly implicated in seemingly unrelated autoimmune, allergic, and malignant diseases (see “Discussion”). The intracellular location of these proteins where they would be inaccessible to circulating antibodies and their lack of disease specificity cast doubt upon their relevance.The best understood example of an antibody-mediated disease is myasthenia gravis with acetylcholine receptor antibodies (for a review, see Ref. 9). Another subgroup of myasthenia gravis patients has antibodies to a muscle-specific tyrosine kinase (MuSK).¹ These antibodies are known to bind to the cell surface and to inhibit the clustering function of MuSK (10). Although the mechanisms of disease are not fully understood, the patients respond to immunotherapies, and the identification of this antigen by a candidate approach has revolutionized the diagnosis and treatment of this subtype of myasthenia (11). In many other conditions, however, no suitable candidate antigens have yet been proposed, limiting the diagnosis and treatment of the disorders.To develop a novel proteomics strategy for identifying cell membrane autoantigens, we used a model system involving antibodies from MuSK antibody-positive patients and from MuSK antibody-negative subjects. We first allowed the antibodies to bind to their target(s) on the intact cell surface, rather than after extraction and denaturation in detergents, so that the antibodies could recognize fully conformational epitopes. The cells, with antibodies already bound, were then solubilized, and the ready formed immune complexes were isolated and either visualized by SDS-PAGE and immunoblotting or identified by mass spectrometry. Although we show the current results as a “proof of principle,” the “conformational membrane antigen isolation and identification” (CMAII) technique could easily be adapted for use in studies of other diseases.     

The cAMP Capture Compound Mass Spectrometry as a Novel Tool for Targeting cAMP-binding Proteins: FROM PROTEIN KINASE A TO POTASSIUM/SODIUM HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CHANNELS

The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics. Capture Compound^TM Mass Spectrometry (CCMS) is a novel technology to address this issue. CCs are trifunctional molecules that accomplish the reversible binding of target protein families to a selectivity group (small molecule), covalent capturing of the bound proteins by photoactivated cross-linking through a reactivity group, and pullout of the small molecule-protein complexes through a sorting function, e.g. biotin. Here we present the design, synthesis, and application of a new Capture Compound to target and identify cAMP-binding proteins in complex protein mixtures. Starting with modest amounts of total protein mixture (65–500 μg), we demonstrate that the cAMP-CCs can be used to isolate bona fide cAMP-binding proteins from lysates of Escherichia coli, mammalian HepG2 cells, and subcellular fractions of mammalian brain, respectively. The identified proteins captured by the cAMP-CCs range from soluble cAMP-binding proteins, such as the catabolite gene activator protein from E. coli and regulatory subunits of protein kinase A from mammalian systems, to cAMP-activated potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels from neuronal membranes and specifically synaptosomal fractions from rat brain. The latter group of proteins has never been identified before in any small molecule protein interaction and mass spectrometry-based proteomics study. Given the modest amount of protein input required, we expect that CCMS using the cAMP-CCs provides a unique tool for profiling cAMP-binding proteins from proteome samples of limited abundance, such as tissue biopsies.cAMP is an important biological second messenger molecule involved in many biological processes, such as adaptation of bacteria to low glucose growing conditions, chemotaxis in slime molds, and various signal transduction processes in metazoa downstream of the activation of hormone receptors (1). The concentration level of cAMP in biological systems is tightly controlled by the activity of adenylyl cyclases that catalyze the formation of cAMP and by the activity of phosphodiesterases, which catalyze the degradation of cAMP. Given the importance of signaling cascades downstream of hormone or neurotransmitter receptors that involve increased formation or degradation of cAMP, the identification and profiling of cAMP effector proteins can be expected to be an essential contribution to elucidate the molecular basis of physiological as well as pathophysiological signaling events.Bona fide effectors of cAMP are proteins that contain a cyclic nucleotide binding domain (CNBD).1 This motif represents a protein domain initially defined and characterized by the crystal structure of the major known cAMP-binding protein from Escherichia coli, the catabolite gene activator protein (2). This domain is present in all known mammalian cAMP-binding proteins as well. Three major classes of proteins exist that contain CNBDs. The first group contains protein kinase A subunits, namely regulatory subunits of protein kinase A isozymes (3), as well as the cGMP-dependent protein kinases (4). A group of Rap guanine nucleotide exchange factors (Epac proteins) that contain CNBDs (5) comprises the second group. Both groups contain key proteins involved in signaling cascades. A number of ion channels that can be directly regulated by cAMP contain CNBDs, such as the cyclic nucleotide-gated channels (6), make up the third group. In particular, potassium/sodium hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role in the pacemaking of heart and brain activity (7). A relatively small number of further proteins that contain CNBDs, such as phosphodiesterase isoforms and a sodium-hydrogen exchange transporter, can be retrieved from searches in databases such as Swiss-Prot.Among the methodological repertoire applied in functional proteomics, small molecule affinity-based techniques seem to be ideal for the task of profiling the cAMP binding proteome subset. Established strategies make use of cAMP affinity beads. These beads comprise cAMP derivatives covalently attached to the polymer backbone via an aminoalkyl linker. The linker may vary in length of the alkyl chain and in the attachment position at the nucleobase (8, 9). This approach, however, suffers from the relatively large amount of protein input required to obtain significant data, precluding e.g. the profiling of the target proteins in samples of limited abundance. Furthermore, it has not been demonstrated yet that affinity-based enrichment of cAMP-binding proteins is suitable for cAMP-binding membrane proteins that are known to be difficult to access. On the other hand, soluble cAMP- and cGMP-binding proteins along with their interaction partners were robustly identified with this methodology. Another approach described in the literature used a cyclic guanosine monophosphate analogue immobilized on a Biacore chip to isolate cGMP- and cAMP-binding proteins from a cell lysate, estimate the quantity of the material, and elute proteins for proteolysis and identification by LC-MS/MS. In addition, for single purified proteins, binding constants can be measured (10). The applicability of this approach to transmembrane cGMP/cAMP-binding proteins, however, has yet to be determined.Here we describe the synthesis and application of a trifunctional Capture Compound^TM (CC) (see Fig. 1A) as a novel approach for the functional isolation of cAMP-binding proteins from complex protein mixtures using low amounts of protein input. In contrast to current pulldown approaches, the CC enables the covalent linkage to the target proteins by a photoactivatable reactivity group in addition to the reversible binding of target proteins by the selectivity group. The Capture Compound-protein conjugate can be isolated from the complex protein mixture via the sorting function (a biotin moiety) of the Capture Compound by means of streptavidin-coated magnetic beads (see Fig. 1, B and C) (11). The cAMP-binding protein-selective Capture Compound described here was successfully applied to the isolation of cAMP-binding proteins from E. coli lysate and cultured eukaryotic HepG2 cells, respectively. Furthermore, we report the applicability of the CCMS approach for the capturing of cAMP-binding HCN channel proteins from rat brain synaptosome preparations as well. To our knowledge, this has not yet been achieved by any cAMP affinity bead approach. In addition, the ion channels, which by antibody- and in situ hybridization-based techniques have been shown to be located in neuronal tissues at synaptic sites (12, 13), have also escaped detection in many detailed proteomics profiling studies conducted to establish the protein complements of synaptic structures (see e.g. Refs. 14–17). Our data suggest that the cAMP-CC approach is uniquely efficient and sensitive for the identification and profiling of cAMP-binding proteins in complex protein mixtures.Open in a separate windowFig. 1.A, schematic design of a CC. Three functionalities are coupled to a core. The selectivity function (red), e.g. modified cAMP, for target recognition; the reactivity function (orange), e.g. diazirines, for covalent cross-linking; and the sorting function (yellow), e.g. biotin, for pullout of captured proteins; and a variable linker (green) that can modify the hydrophilicity of the system are shown. B, structure of 8-AHA-cAMP-CC (7c), which represents one of several cAMP-CCs that are available. C, flow chart of the CCMS technology.     

Towards a phylogeny and evolution of Acochlidia (Mollusca: Gastropoda: Opisthobranchia)

The Acochlidia are unique among opisthobranch gastropods in combining extremely high morphological and ecological diversity with modest species diversity. The phylogeny of acochlidians has never been addressed by cladistic means, as their evolution has remained unknown. This study gives a first overview on more than 150 biological and morphological characters that are potentially useful for phylogenetic analysis. Based on 107 characters, a parsimony analysis (PAUP) was performed for all 27 valid acochlidian species together with 11 (plus two) outgroup taxa. The resulting strict consensus tree shows a moderate overall resolution, with at least some bootstrap support for most resolved nodes. The Acochlidia are clearly monophyletic, and originate from an unresolved basal opisthobranch level. The Acochlidia split into the Hedylopsacea (Tantulum (Hedylopsis (Pseudunela (Strubellia (‘Acochlidium’, ‘Palliohedyle’))))) and Microhedylacea (Asperspina (Pontohedyle, ‘Parhedyle’, ‘Microhedyle’, (Ganitus, Paraganitus))). The formerly enigmatic Ganitidae, resembling sacoglossan opisthobranchs by having dagger‐like rachidian radular teeth, are likely to be highly derived microhedylids. The paraphyly of some of the traditionally recognized family level taxa induced a preliminary reclassification. From the phylogenetic hypothesis obtained, we conclude that the acochlidian ancestor was marine mesopsammic. The colonization of limnic systems occurred twice, independently: first in the Caribbean (with the development of the small interstitial Tantulum elegans), and second in the Indo‐Pacific, with a radiation of large‐sized benthic acochlidian species. The evolution of extraordinary reproductive features, such as hypodermic impregnation by a complex copulative aparatus in hedylopsaceans, cutaneous insemination via spermatophores in microhedylaceans, and gonochorism in Microhedylidae s.l. (including Ganitidae), is discussed. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 124–154.     

Clostridium difficile toxins: more than mere inhibitors of Rho proteins

Toxin A (TcdA) and Toxin B (TcdB) are the major pathogenicity factors of the Clostridium difficile-associated diarrhoea (CDAD). The single-chained protein toxins enter their target cells by receptor-mediated endocytosis. New data show the critical role of auto-catalytic processing for target cell entry. Inside the cell, the toxins mono-glucosylate and thereby inactivate low molecular mass GTP-binding proteins of the Rho subfamily. Toxin-treated cells respond to RhoA glucosylation with up-regulation and activation of the pro-apoptotic Rho family protein RhoB. These data reinforce the critical role of the glucosyltransferase activity for programmed cell death and show that TcdA and TcdB, generally classified as broad-spectrum inhibitors of Rho proteins, are also capable of activating Rho proteins.