Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion‐competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13‐4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13‐1 and Munc13‐4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13‐4‐deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13‐4 and Munc13‐1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13‐1 and Munc13‐4, are functionally redundant in murine CTLs .      

High resolution crystal structures of Mycobacterium tuberculosis adenosine kinase: insights into the mechanism and specificity of this novel prokaryotic enzyme

Adenosine kinase (ADK) catalyzes the phosphorylation of adenosine (Ado) to adenosine monophosphate (AMP). It is part of the purine salvage pathway that has been identified only in eukaryotes, with the single exception of Mycobacterium spp. Whereas it is not clear if Mycobacterium tuberculosis (Mtb) ADK is essential, it has been shown that the enzyme can selectively phosphorylate nucleoside analogs to produce products toxic to the cell. We have determined the crystal structure of Mtb ADK unliganded as well as ligand (Ado) bound at 1.5- and 1.9-A resolution, respectively. The structure of the binary complexes with the inhibitor 2-fluoroadenosine (F-Ado) bound and with the adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (non-hydrolyzable ATP analog) bound were also solved at 1.9-A resolution. These four structures indicate that Mtb ADK is a dimer formed by an extended beta sheet. The active site of the unliganded ADK is in an open conformation, and upon Ado binding a lid domain of the protein undergoes a large conformation change to close the active site. In the closed conformation, the lid forms direct interactions with the substrate and residues of the active site. Interestingly, AMP-PCP binding alone was not sufficient to produce the closed state of the enzyme. The binding mode of F-Ado was characterized to illustrate the role of additional non-bonding interactions in Mtb ADK compared with human ADK.     

Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.     

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid‐mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.     

Merkel cell carcinoma in a malignant pleural effusion: case report

BACKGROUND: Merkel cell (neuroendocrine) carcinoma is a small round blue cell malignant neoplasm that primarily presents in the skin. The diagnosis of Merkel cell carcinoma in a pleural fluid is challenging because of the morphological similarity to many other malignant neoplasms. Immunohistochemical stains can be essential to establish the diagnosis of Merkel cell carcinoma. CASE PRESENTATION: A 77 year-old woman presented with a mass in her right buttock thought clinically to be a boil or sebaceous cyst. Upon histopathologic review including immunohistochemical analysis, a diagnosis of Merkel cell carcinoma was rendered. Wide-excision and sentinel lymph node biopsy revealed negative margins and no evidence of metastasis. Ten months later she complained of bone pain and a bone scan revealed multiple lesions. An abdominal CT scan revealed a T4 vertebral mass and local radiotherapy was administered. Two months later the patient presented with shortness of breath. A chest radiograph showed an effusion and thoracentesis was performed. The fluid was confirmed to contain metastatic Merkel cell carcinoma by cytology and immunohistochemical analysis. CONCLUSIONS: Merkel cell carcinoma is an aggressive neoplasm that can, despite careful surgical management, occasionally present as a malignant pleural effusion in a relatively short time period. Immunohistochemical analysis can aid in confirming this rare outcome.     

MicroRNA function and neurotrophin BDNF

MicroRNAs (miRs), endogenous small RNAs, regulate gene expression through repression of translational activity after binding to target mRNAs. miRs are involved in various cellular processes including differentiation, metabolism, and apoptosis. Furthermore, possible involvement of miRs in neuronal function have been proposed. For example, miR-132 is closely related to neuronal outgrowth while miR-134 plays a role in postsynaptic regulation, suggesting that brain-specific miRs are critical for synaptic plasticity. On the other hand, numerous studies indicate that BDNF (brain-derived neurotrophic factor), one of the neurotrophins, is essential for a variety of neuronal aspects such as cell differentiation, survival, and synaptic plasticity in the central nervous system (CNS). Interestingly, recent studies, including ours, suggest that BDNF exerts its beneficial effects on CNS neurons via up-regulation of miR-132. Here, we present a broad overview of the current knowledge concerning the association between neurotrophins and various miRs.     

Dark septate endophyte and arbuscular mycorrhizal status of vegetation colonizing a bottomland hardwood forest after a 100 year flood

Levels of arbuscular mycorrhizal (AM) colonization and dark septate endophyte (DSE) colonization were assessed in the vegetation recolonizing a remnant bottomland hardwood forest in north central Texas following a 100 year flood. Thirty seven plant species representing 21 dicotyledonous and 2 montocotyledonous families established following floodwater recession. AM and/or DSE were found in all species. AM colonization was found in 31 out of the 37 species assessed including both monocotyledonous families (Poaceae and Cyperaceae) and 17 out of 21 dicotyledonous families (Acanthaceae, Asteraceae, Boraginaceae, Cucurbitaceae, Euphorbiaceae, Lamiaceae, Loganiaceae, Lythraceae, Malvaceae, Onagraceae, Pedaliaceae, Ranunculaceae, Sapindaceae, Scrophulariaceae, Solanaceae, Verbanaceae and Violaceae). DSE were found in 31 out of 37 species assessed including both monocotyledonous families and 15 out of 21 dicotyledonous families (Amaranthaceae, Asteraceae, Boraginaceae, Brassicaceae, Cucurbitaceae, Euphorbiaceae, Lamiaceae, Lythraceae, Malvaceae, Pedaliaceae, Phytolaccaceae, Polygonaceae, Ranunculaceae, Sapindaceae, Scrophulariaceae and Violaceae). There were no detectable differences in AM or DSE colonization levels among wetland indicator groups (p > 0.05). Levels of DSE colonization were negatively correlated with vesicular colonization and hyphal colonization for the obligate wetland species. There were no other significant relationships between AM and DSE colonization detected. Spearman rank order correlation coefficients did not differ significantly among wetland indicatory category for any level of AM or DSE colonization.