Selective delivery of 2-hydroxy APA to Trypanosoma brucei using the melamine motif

Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of selectively delivering this compound to the parasite. The best compound described here shows an increased in vitro trypanocidal activity compared with the parent.     

NMR secondary structure and interactions of recombinant human MOZART1 protein,a component of the gamma‐tubulin complex

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.     

Characterization of the Synechocystis Strain PCC 6803 Penicillin-Binding Proteins and Cytokinetic Proteins FtsQ and FtsW and Their Network of Interactions with ZipN

Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.The peptidoglycan layer (PG) of bacterial cell wall is a major determinant of cell shape, and the target of our best antibiotics. It is built from long glycan strands of repeating disaccharides cross-linked by short peptides (38). The resultant meshwork structure forms a strong and elastic exoskeleton essential for maintaining shape and withstanding intracellular pressure. Cell morphogenesis and division have been essentially studied in the rod-shaped organisms Escherichia coli and Bacillus subtilis, which divide through a single medial plane (8, 10, 21, 23). These organisms have two modes of cell wall synthesis: one involved in cell elongation and the second operating in septation (2). Each mode of synthesis is ensured by specific protein complexes involving factors implicated in the last step of PG synthesis (2). The complete assembly of PG requires a glycosyl transferase that polymerizes the glycan strands and a transpeptidase that cross-links them via their peptide side chains (35). Both activities are catalyzed by penicillin-binding proteins (PBPs), which can be divided into three classes: class A and class B high-molecular-weight (HMW) PBPs and class C low-molecular-weight (LMW) PBPs (35).Class A PBPs exhibit both transglycosylase and transpeptidase activities. In E. coli, they seem to be nonspecialized (2), as they operate in the synthesis of both cylindrical wall (cell elongation) and septal PG (cytokinesis). In B. subtilis, PBP1 (class A) is partially localized to septal sites and its depletion leads to cell division defects (31).Class B PBPs, which comprise two proteins in most bacteria, are monofunctional transpeptidases (35), each involved in longitudinal and septal growth of cell wall, respectively (36). In E. coli, this protein, PBP3, is also termed FtsI, because it belongs to the Fts group of cell division factors whose depletion leads to the filamentation phenotype (11). These at least 10 Fts proteins are recruited to the division site at mid-cell in the following sequential order: FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL/FtsB, FtsW, FtsI, and FtsN (11). The cytoplasmic protein FtsZ is the first recruited to the division site, where it polymerizes in a ring-like structure (1), which serves as a scaffold for the recruitment of the other Fts proteins and has been proposed to drive the division process (6). Together the Fts proteins form a complex machine coordinating nucleoid segregation, membrane constriction, septal PG synthesis, and possibly membrane fusion.Unlike the other PBPs, class C PBPs do not operate in PG synthesis but rather in maturation or recycling of PG during cell septation (35). They are subdivided into four types. Class C type 5 PBP removes the terminal d-alanine residue from pentapeptide side-chains (dd-carboxypeptidase activity). Types 4 and 7 are able to cleave the peptide cross-links (endopeptidase activity). Finally, type AmpH, which does not have a defined enzymatic activity, is believed to play a role in the normal course of PG synthesis, remodeling or recycling (for a review, see reference 35).In contrast to rod-shaped bacteria, less is known concerning PG synthesis, morphogenesis, and cytokinesis, and their relationships, in spherical-celled bacteria, even though a wealth of them have a strong impact on the environment and/or human health. Furthermore, unlike rod-shaped bacteria spherical-celled bacteria possess an infinite number of potential division planes at the point of greater cell diameter, and they divide through alternative perpendicular planes (26, 36, 37, 39). The spherical cells of Staphylococcus aureus seem to insert new PG strands only at the septum, and accordingly the unique class A PBP localizes at the septum during cell division (36). In contrast, the rugby-ball-shaped cells of Streptococcus pneumoniae synthesize cell wall at both the septum and the neighboring region called “equatorial rings” (36). Accordingly, class A PBP2a and PBP1a were found to operate in elongation and septation, respectively (29).In cyanobacteria, which are crucial to the biosphere in using solar energy to renew the oxygenic atmosphere and which make up the biomass for the food chain (7, 30, 40), cell division is currently investigated in two unicellular models with different morphologies: the rod-shaped Synechococcus elongatus strain PCC 7942 (19, 28) and the spherical-celled Synechocystis strain PCC 6803 (26), which both possess a small fully sequenced genome (http://genome.kazusa.or.jp/cyanobase/) that is easily manipulable (18). In both organisms FtsZ and ZipN/Arc6, a protein occurring only in cyanobacteria (ZipN) and plant chloroplasts (Arc6), were found to be crucial for cytokinesis (19, 26, 28) and to physically interact with each other (25, 26). Also, interestingly, recent studies of cell division in the filamentous cyanobacterium Anabaena (Nostoc) strain PCC 7120, showed that this process is connected with the differentiation of heterocysts, the cells dedicated to nitrogen fixation (34).In a continuous effort to study the cell division machine of the unicellular spherical cyanobacterium Synechocystis, we have presently characterized its eight presumptive PBPs (22) that define three classes and the putative cytokinetic proteins FtsQ and FtsW, as well as their network of interactions between each other and ZipN. Both FtsI and FtsQ were found to be key players in cell division in interacting with ZipN and class A PBPs. Consequently, ZipN in interacting with FtsZ (26), FtsI, and FtQ, like the FtsA protein of E. coli, could play a role similar to FtsA, which is absent in cyanobacteria and chloroplasts.     

Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE

Using a bacterial two-hybrid system and a combination of in vivo and in vitro assays that take advantage of the green fluorescent reporter protein (GFP), we have investigated the localization and the protein-protein interaction of several key components of the cytokinetic machinery of cyanobacteria (i.e. the progenitor of chloroplast). We demonstrate that (i) the ftsZ and zipN genes are essential for the viability of the model cyanobacterium Synechocystis sp. PCC 6803, whereas the minCDE cluster is dispensable for cell growth; (ii) the GTP-binding domain of FtsZ is crucial to FtsZ assembly into the septal ring at mid-cell; (iii) the Z-ring of deeply constricted daughter cells is oriented perpendicularly to the mother Z-ring, showing that Synechocystis divides in alternating perpendicular planes; (iv) the MinCDE system affects the morphology of the cell, as well as the position and the shape of FtsZ structures; and (v) MinD is targeted to cell membranes in a process involving its C-terminal amphipathic helix, but not its ATP-binding region. Finally, we have also characterized a novel Z-interacting protein, ZipN, the N-terminal DnaJ domain of which is critical to the decoration of the Z-ring, and we report that this process is independent of MinCDE.     

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.     

Numb Localizes at Endosomes and Controls the Endosomal Sorting of Notch after Asymmetric Division in Drosophila

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Highlights? Numb relocalizes from the cortex to sorting endosomes at cytokinesis ? Numb is not essential for the internalization of Notch and Sanpodo ? Numb inhibits the recycling of Notch and Sanpodo ? Recycling inhibition in one daughter cell imposes directionality to Notch signaling     

Pure and mixed mucinous carcinoma of the breast: fine needle aspiration cytology findings and review of the literature

J. Cyrta, F. Andreiuolo, S. Azoulay, C. Balleyguier, C. Bourgier, C. Mazouni, M.‐C. Mathieu, S. Delaloge and P. Vielh
Pure and mixed mucinous carcinoma of the breast: fine needle aspiration cytology findings and review of the literature Objective: Mucinous (colloid) breast carcinoma accounts for 1–6% of all breast cancer. It comprises pure mucinous tumours and mixed infiltrating ductal carcinomas with a mucinous component. As this latter mixed form has a worse prognosis than pure colloid carcinoma, making this diagnosis on fine needle aspiration cytology (FNAC) might influence the choice of treatment. Methods: We report a consecutive series of 22 cases consisting of 17 mixed and five pure mucinous carcinomas diagnosed by cytology and verified on histopathology. Patients underwent FNAC at the one‐stop clinic of our institution during a 7‐year period of time. Cytological findings were evaluated by a semi‐quantitative method and included percentage of smear surface occupied by mucin, shape of cell groupings, size and outline of tumour nuclei as well as presence or absence of nucleolus. Results: Three of five pure mucinous carcinomas displayed at least two of the following features: abundant mucin, small nuclei and/or regular nuclear outlines. Sparse mucin, large nuclei, irregular nuclear outlines or the presence of nucleoli were found in 7 out of 17 mixed mucinous carcinomas but not in pure tumours. Conclusion: Cytopathological identification of patients with pure mucinous carcinomas may be performed in a limited number of cases.