Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites

Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for α2–3- over α2–6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to α2–9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6′sulfo-sialyl Lewis^x might have implications for tissue tropism.     

PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas

Targeted intervention of the B-Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next-generation inhibitor with exquisite specificity against the V600E oncogene and striking anti-melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E-positive cells in a variety of in vitro experimental systems; follow-up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.     

Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase,and its spatial and temporal changes during leaf development

The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.     

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.     

Patterns of protein synthesis in the chick blastula: A comparison of the component areas of the epiblast and the primary hypoblast

The patterns of protein synthesis are examined in the hypoblast and in the areas that comprise the epiblast, that is, the area opaca, the marginal zone, and the central area, during the blastula stage which marks the beginning of the interaction between the epiblast and hypoblast for induction of the primitive streak. The results demonstrate that there are distinct qualitative and quantitative differences in protein patterns in individual areas of blastoderm, the differences being most distinct between the hypoblast and any of the component areas of the epiblast. These differences in patterns of proteins suggest that the component areas of the chick blastula have already diverged to different developmental fates before any apparent morphogenetic differentiation, that is, the appearance of the primitive streak.     

Origin and evolution of the Ig-like domains present in mammalian leukocyte receptors: insights from chicken, frog, and fish homologues

In mammals many natural killer (NK) cell receptors, encoded by the leukocyte receptor complex (LRC), regulate the cytotoxic activity of NK cells and provide protection against virus-infected and tumor cells. To investigate the origin of the Ig-like domains encoded by the LRC genes, a subset of C2-type Ig-like domain sequences was compiled from mammals, birds, amphibians, and fish. Phylogenetic analysis of these sequences generated seven monophyletic groups in mammals (MI, MII, and FcI, FcIIa, FcIIb, FcIII, FcIV), two in chicken (CI, CII), four in frog (FI–FIV), and five in zebrafish (ZI–ZV). The analysis of the major groups supported the following order of divergence: ZI [or a common ancestor of ZI and F (a cluster composed of the FcIII and FIII groups)], F, CII (or a common ancestor of CII and MII), MII, and MI–CI. The relationships of the remaining groups were unclear, since the phylogenetic positions of these groups were not supported by high bootstrap values. Two main conclusions can be drawn from this analysis. First, the two groups of mammalian LRC sequences must diverged before the separation of the avian and mammalian lineages. Second, the mammalian LRC sequences are most closely related to the Fc receptor sequences and these two groups diverged before the separation of birds and mammals.Electronic Supplementary Material Supplementary material is available for this article at     

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far–including the gold standard CellSearch—rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAM^low/neg cell line and EpCAM^neg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAM^pos (e.g. MCF7, SKBR3) and EpCAM^low/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAM^neg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAM^pos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAM^low/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAM^low/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAM^neg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAM^neg CTCs could be identified [range of 1–24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAM^neg dual-positive (CK^pos/CD45^pos) cells could be traced in 28 out of 29 samples [range 1–480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAM^neg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAM^neg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.