Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

Synthesis of highly potent and selective hetaryl ureas as integrin alpha(V)beta3-receptor antagonists

Solid-phase synthesis and SAR of integrin alpha(V)beta3-receptor antagonists containing a urea moiety as non-basic guanidine mimetic are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta3 in the nanomolar range and high selectivity versus related integrins like alpha(IIb)beta3. For selected examples efficacy in functional cellular assays is demonstrated.     

Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 alpha-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).     

Quantitative Site-specific Phosphorylation Dynamics of Human Protein Kinases during Mitotic Progression

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.Reversible phosphorylation is a ubiquitous posttranslational protein modification that is involved in the regulation of almost all biological processes (1–3). In human, 518 protein kinases have been identified in the genome that phosphorylate the majority of cellular proteins and increase the diversity of the proteome by severalfold (4). Addition of a phosphate group to a protein can alter its structural, catalytic, and functional properties; hence, kinases require tight regulation to avoid unspecific phosphorylation, which can be deleterious to cells (5–7). As a result, cells use a variety of mechanisms to ensure proper regulation of kinase activities (8). Importantly, most kinases are also in turn regulated through autophosphorylation and phosphorylation by other kinases, thus generating complex phosphorylation networks. In particular, phosphorylation on activation segments is a common mechanism to modulate kinase activities (9–11), but additional phosphorylation sites are also frequently required for fine tuning of kinase localizations and functions (12). Some kinases contain phosphopeptide binding domains that recognize prephosphorylated sites on other kinases, resulting in processive phosphorylation and/or targeting of kinases to distinct cellular locations (13–16). Because such priming phosphorylation events depend on the activities of the priming kinases, these motifs act as conditional docking sites and restrict the interaction with docking kinases to a particular point in time and physiological state. In addition, phosphorylation sites may act through combinatorial mechanisms or through cross-talk with other posttranslational modifications (PTMs)¹ (17, 18), thus further increasing the complexity of kinase regulatory networks.Regulation of kinases is of particular interest in mitosis as most of the mitotic events are regulated by reversible protein phosphorylation (19). During mitosis, error-free segregation of sister chromatids into the two daughter cells is essential to ensure genomic stability. Physically, this process is carried out by the mitotic spindle, a highly dynamic microtubule-based structure. After entry into mitosis, the major microtubule-organizing centers in animal cells, the centrosomes, start to increase microtubule nucleation and move to opposite poles of the cell. Throughout prometaphase, microtubules emanating from centrosomes are captured by kinetochores, protein complexes assembled on centromeric chromosomal DNA. This eventually leads to the alignment of all chromosomes in a metaphase plate. Because proper bipolar attachment of chromosomes to spindle microtubules is essential for the correct segregation of chromosomes, this critical step is monitored by a signaling pathway known as the spindle assembly checkpoint (SAC) (20). This checkpoint is silenced only after all chromosomes have attached to the spindle in a bioriented fashion, resulting in the synchronous segregation of sister chromatids during anaphase. Simultaneously, a so-called central spindle is formed between the separating chromatids, and the formation of a contractile ring initiates cytokinesis. Finally, in telophase, the chromosomes decondense and reassemble into nuclei, whereas remnants of the central spindle form the midbody, marking the site of abscission. Cyclin-dependent kinase 1 (Cdk1), an evolutionarily conserved master mitotic kinase, is activated prior to mitosis and initiates most of the mitotic events. Cdk1 works in close association with other essential mitotic kinases such as Plk1, Aurora A, and Aurora B for the regulation of mitotic progression (19, 21–24). Plk1 and Aurora kinases dynamically localize to different subcellular locations to perform multiple functions during mitosis and are phosphorylated at several conserved sites. Although little is known about the precise roles of these phosphorylation sites, emerging data indicate that they are involved in regulating localization-specific functions (25, 26). Furthermore, the kinases Bub1, BubR1, and TTK (Mps1) and kinases of the Nek family play important roles in maintaining the fidelity and robustness of mitosis (19). Recently, a genome-wide RNA-mediated interference screen identified M phase phenotypes for many kinases that have not previously been implicated in cell cycle functions, indicating that additional kinases have important mitotic functions (27).Although protein phosphorylation plays a pivotal role in the regulation of cellular networks, many phosphorylation events remain undiscovered mainly because of technical limitations (28). The advent of mass spectrometry-based proteomics along with developments in phosphopeptide enrichment methods has enabled large scale global phosphoproteomics studies (29, 30). However, the number of phosphorylation sites identified on kinases is limited compared with other proteins because of their frequently low expression levels. To overcome this problem, small inhibitor-based kinase enrichment strategies were developed, resulting in the identification of more than 200 kinases from HeLa cell lysates (31, 32). This method was also used recently to compare the phosphokinomes during S phase and M phase of the cell cycle, resulting in the identification of several hundreds of M phase-specific kinase phosphorylation sites (31). In the present study, we address the dynamics of the phosphokinome during mitotic progression using large scale cell synchronization at three distinct mitotic stages, small inhibitor-based kinase enrichment, and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry. Thus, we determined the mitotic phosphorylation dynamics of more than 900 kinase phosphorylation sites and identified distinctly regulated kinase interaction networks. Our results provide a valuable resource for the dynamics of the kinome during mitotic progression and give insight into the system properties of kinase interaction networks.     

Grey and White Matter Changes across the Amyotrophic Lateral Sclerosis-Frontotemporal Dementia Continuum

There is increasing evidence that amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) lie on a clinical, pathological and genetic continuum with patients of one disease exhibiting features of the other. Nevertheless, to date, the underlying grey matter and white matter changes across the ALS-FTD disease continuum have not been explored. In this study fifty-three participants with ALS (n = 10), ALS-FTD (n = 10) and behavioural variant FTD (bvFTD; n = 15) as well as controls (n = 18), underwent detailed clinical assessment plus structural imaging using voxel-based morphometry (VBM) and diffusion tensor imaging (DTI) analysis of magnetic resonance brain imaging to examine grey and white matter differences and commonalities across the continuum. Importantly, patient groups were matched for age, education, gender and disease duration. VBM and DTI results showed that changes in the ALS group were confined mainly to the motor cortex and anterior cingulate as well as their underlying white matter tracts. ALS-FTD and bvFTD showed widespread grey matter and white matter changes involving frontal and temporal lobes. Extensive prefrontal cortex changes emerged as a marker for bvFTD compared to other subtypes, while ALS-FTD could be distinguished from ALS by additional temporal lobe grey and white matter changes. Finally, ALS could be mainly distinguished from the other two groups by corticospinal tract degeneration. The present study shows for the first time that FTD and ALS overlap in anterior cingulate, motor cortex and related white matter tract changes across the whole continuum. Nevertheless, frontal and temporal atrophy as well as corticospinal tract degeneration emerged as marker for subtype classification, which will inform future diagnosis and target disease management across the continuum.     

Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

Background  

Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.