Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP₂ and PIP₃ molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110β deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110β and the more subtle role of p110α in the production of PIP₃ molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP₂ during stimulation, opening new perspectives in phosphoinositide signaling in platelets.     

Microtubule assembly and disassembly at alkaline pH

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.     

Localization of short/branched chain Acyl-CoA dehydrogenase (ACADSB) to human chromosome 10

Short/branched chain acyl-CoA dehydrogenase, SBCAD (gene symbol ACADSB), is a member of the acyl-CoA dehydrogenase family of genes with activity toward the short/branched chain acyl-CoA derivatives as well as short/straight chain acyl-CoAs. Southern blot analysis of DNA from a panel of human/rodent somatic cell hybrids localized ACADSB to human chromosome 10, and fluorescence in situ hybridization experiments confirmed the chromosomal assignment and refined the subchromosomal localization to 10q25–q26.     

Isoforms of the human PDZ-73 protein exhibit differential tissue expression

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.     

Intercontinental distribution of Plagiochila corrugata (Plagiochilaceae, Hepaticae) inferred from nrDNA ITS sequences and morphology

Plagiochila sect. Vagae is a large pantropical clade that is characterized morphologically by frequent terminal branching, vegetative distribution by propagules on the ventral surface of the leaves and a capsule wall with thickenings in all layers. Plagiochila corrugata from Brazil is characterized by strongly undulate, toothed leaf margins and represents the only known neotropical species of sect. Vagae with unispiral elaters. Plagiochila cambuena from Madagascar is distinguished by the same features. Maximum likelihood and parsimony analyses of 38 nrDNA ITS sequences of Plagiochila reveal P. corrugata and P. cambuena in a weakly (ML) to well (MP) supported monophyletic lineage within P.  sect.  Vagae . As an outcome of the morphological and molecular investigation, P. cambuena is relegated to the synonymy of P. corrugata. Plagiochila corrugata is placed in a Vagae -subclade with 11 further American species. The range of P. corrugata can be ascribed to long-range dispersal from the Neotropics rather than a Gondwanan distribution. Species from tropical Asia and Africa are placed at the base of the Vagae clade. Branch length within P.  sect.  Vagae points to a sudden radiation.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 469–481.     

Tube-Like Structures with Co-Expression of D2-40 and CD34: Newly Formed Vasculatures?

Background: A great number of in vitro and in vivo studies have suggested that many pathways or factors can stimulate angiogenesis and lymphangiogenesis, which facilitate tumor progression and metastasis. However, the morphological and immunohistochemical profile of newly formed vasculatures has not been elucidated, making it difficult to differentiate them from the pre-existing ones, and to identify their unique molecular profiles for diagnosis and therapeutic interventions.Experimental findings: As cytokeratin (CK)-19 is a well-recognized stem cell marker and CK-19-positive cells are frequently detected in the peripheral blood of patients with metastatic cancer, our recent studies have assessed the involvement of CK-19 in the formation of new vasculatures in primary colorectal cancer (CRC) tissues. Our studies showed that a subset of lymph node-positive cases harbored some isolated normal epithelial structures with distinct CK-19 immunostaining within an otherwise CK-19-negative background. These structures are exclusively located within or adjacent to lymphoid follicles and are often surrounded by tube-like structures expressing lymphatic endothelial marker D2-40. Similar structures are more frequently seen at the junctions between pre-invasive and invasive CRC with the following features: (1). they consist of a single layer of endothelial cells that express both D2-40 and CD34, (2). their endothelial walls are often incomplete with disseminated cells protruding into the adjacent stroma, and (3). they are exclusively associated with disseminated CK-19-positive cellsHypothesis: Based on these findings, we propose that these tube-like structures represent newly formed vasculatures, which are derived by the convergence of aberrant lymphocyte infiltration and tumor stem cells. Because of their close physical proximity, tumor stem cells within the epithelial and stromal components contribute equally and coordinately to the morphogenesis of new vasculatures, which constitutes the basis for the unique morphologic and immunohistochemical features of newly formed vasculatures. Our hypothesis appears to be applicable to all epithelium-derived cancers.     

HSPVdb—the Human Short Peptide Variation Database for improved mass spectrometry-based detection of polymorphic HLA-ligands

T cell epitopes derived from polymorphic proteins or from proteins encoded by alternative reading frames (ARFs) play an important role in (tumor) immunology. Identification of these peptides is successfully performed with mass spectrometry. In a mass spectrometry-based approach, the recorded tandem mass spectra are matched against hypothetical spectra generated from known protein sequence databases. Commonly used protein databases contain a minimal level of redundancy, and thus, are not suitable data sources for searching polymorphic T cell epitopes, either in normal or ARFs. At the same time, however, these databases contain much non-polymorphic sequence information, thereby complicating the matching of recorded and theoretical spectra, and increasing the potential for finding false positives. Therefore, we created a database with peptides from ARFs and peptide variation arising from single nucleotide polymorphisms (SNPs). It is based on the human mRNA sequences from the well-annotated reference sequence (RefSeq) database and associated variation information derived from the Single Nucleotide Polymorphism Database (dbSNP). In this process, we removed all non-polymorphic information. Investigation of the frequency of SNPs in the dbSNP revealed that many SNPs are non-polymorphic “SNPs”. Therefore, we removed those from our dedicated database, and this resulted in a comprehensive high quality database, which we coined the Human Short Peptide Variation Database (HSPVdb). The value of our HSPVdb is shown by identification of the majority of published polymorphic SNP- and/or ARF-derived epitopes from a mass spectrometry-based proteomics workflow, and by a large variety of polymorphic peptides identified as potential T cell epitopes in the HLA-ligandome presented by the Epstein–Barr virus cells.     

Temporal variability in the spatial and environmental determinants of functional metacommunity organization – stream fish in a human‐modified landscape

1. Quantifying the relative importance of environmental filtering versus regional spatial structuring has become an intensively studied area in the context of metacommunity ecology. However, most studies have evaluated the role of environmental and spatial processes using taxonomic data sets of single snapshot surveys. 2. Here, we examined temporal changes in patterns and possible processes behind the functional metacommunity organization of stream fishes in a human‐modified landscape. Specifically, we (i) studied general changes in the functional composition of fish assemblages among 40 wadeable stream sites during a 3‐year study period in the catchment area of Lake Balaton, Hungary, (ii) quantified the relative importance of spatial and environmental factors as determinants of metacommunity structure and (iii) examined temporal variability in the relative role of spatial and environmental processes for this metacommunity. 3. Partial triadic analysis showed that assemblages could be effectively ordered along a functional gradient from invertebrate consuming species dominated by the opportunistic life‐history strategy, to assemblages with a diverse array of functional attributes. The analysis also revealed that functional fish assemblage structure was moderately stable among the sites between the sampling periods. 4. Despite moderate stability, variance partitioning using redundancy analyses (RDA) showed considerable temporal variability in the contribution of environmental and spatial factors to this pattern. The analyses also showed that environmental variables were, in general, more important than spatial ones in determining metacommunity structure. Of these, natural environmental variables (e.g. altitude, velocity) proved to be more influential than human‐related effects (e.g. pond area, % inhabited area above the site, nutrient enrichment), even in this landscape with relatively low variation in altitude and stream size. 5. Pond area was, however, the most important human stressor variable that was positively associated with the abundance of non‐native species with diverse functional attributes. The temporal variability in the relative importance of environmental and spatial factors was probably shaped by the release of non‐native fish from fish ponds to the stream system during flood events. 6. To conclude, both spatial processes and environmental control shape the functional metacommunity organization of stream fish assemblages in human‐modified landscapes, but their importance can vary in time. We argue, therefore, that metacommunity studies should better consider temporal variability in the ecological mechanisms (e.g. dispersal limitation, species sorting) that determine the dynamics of landscape‐level community organization.     

Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.