Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.     

The 1.6 ? Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.     

The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction.

G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

Isoform-specific O-Glycosylation of Osteopontin and Bone Sialoprotein by Polypeptide N-Acetylgalactosaminyltransferase-1

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.     

Relationships between the flexed-arm hang and select measures of muscular fitness

The flexed-arm hang (FAH) has been used to assess arm and shoulder girdle strength for 35 years despite little evidence to support its use. The purpose of this study was to determine what muscular fitness component, if any, was related to the FAH. The traditional overgrip chin-above-bar test and 5 different variations were compared with absolute strength (1 repetition maximum [1RM] lat pull down), relative strength (1RM.mass(-1)), and muscle endurance (repetitions to failure at 70% of the 1RM). Sixty college-age women volunteered for the study. Relationships were examined using Pearson Product Moment Correlation. No significant relationship was found between any of the FAH variations and absolute strength or muscle endurance; however, all FAH variations correlated significantly with relative strength (1RM.mass(-1)). The strongest relationship was with the undergrip FAH timed to 90 degrees of elbow extension (r = 0.72). Investigators concluded that the FAH is a test of weight-relative muscular strength and appears unrelated to absolute strength or muscle endurance.