Formaldehyde-amine fixatives for immunocytochemistry of cultured Xenopus myocytes

Although formaldehyde is commonly used in immunocytochemical studies, this fixative can cause distortions in cell structure. We tested the possibility that adducts of formaldehyde and primary amines could be used as improved fixatives for immunolabeling studies of cultured cells. A variety of primary amines were reacted with formaldehyde and applied to cultured Xenopus muscle cells, after which the cultures were labeled for immunofluorescence. Amine-formaldehyde fixatives improved structural preservation of the myocytes as compared with formaldehyde alone. The extent of improvement depended on the amine tested; the best results were obtained using cyclohexylamine. Immunofluorescence localization of a variety of antigens was better in myocytes fixed with cyclohexylamine-formaldehyde than in cells fixed with formaldehyde alone. In addition, the fixative provided good ultrastructural preservation of cytoskeletal structures and permitted immunogold labeling for alpha-actinin by use of pre-embedding labeling techniques.     

Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response.     

Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD], 20 microM) and low-affinity (KD, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.     

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.     

Lysophosphatidic acid acyltransferase β (LPAATβ) promotes the tumor growth of human osteosarcoma

Background

Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.

Methodology/Principal Findings

Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.

Conclusions/Significance

Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors.     

Segregation of the classical transmitters norepinephrine and acetylcholine and the neuropeptide Y in sympathetic neurons: Modulation by ciliary neurotrophic factor or prolonged growth in culture

Recent evidence has demonstrated that cotransmission from mammalian neurons is not uniquely achieved by costorage and corelease of transmitters and cotransmitters from single varicosities, but also by the concurrent release of mediators segregated in separate synapses of individual neurons. An important question to be addressed is whether neurons show defined patterns of segregation or whether this is a plastic feature. We addressed this question by exploring the segregation pattern of the classical sympathetic transmitters norepinephrine (NE) and acetylcholine (ACh) and the cotransmitter neuropeptide Y (NPY) in sympathetic ganglionic neurons cocultured with cardiac myocytes. Using antibodies against NPY and the vesicular NE and ACh transporters VMAT2 and vesicular acetylcholine transporter (VAChT), we investigated the effect of ciliary neurotrophic factor (CNTF) or long (three weeks) culture periods on the segregation of VMAT2, VAChT, and NPY to separate varicosities. We found that although ganglionic neurons showed cell body coexpression of all the markers examined after three days, VMAT2 was segregated from VAChT in 43% of the VAChT‐positive varicosities. In contrast, VMAT2 was only segregated from NPY in 16.3% of the NPY‐positive varicosities. Cotransmitter segregation and VAChT expression was potentiated by both CNTF and longer times in culture. We also found two types of varicosities: one was smaller and located further from neuronal somata, and the other was larger, proximal to neuronal somata and had a higher level of segregation. These data demonstrate segregation of classical transmitters in sympathetic neurons and plasticity of neurotransmitter segregation. Finally, we discuss a possible functional correlate of segregation in sympathetic neurons. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 913–928, 2010