Sound radiation from randomly vibrating beams of finite circular cross section

The radiation of sound from vibrating cylindrical beams is analyzed based on the frequency of the beam vibrations and the physical characteristics of the beam and its surroundings. A statistical analysis of random beam vibrations allows this result to be independent of the boundary conditions at the ends of the beam. The acoustic power radiated by the beam can be determined from a knowledge of the frequency band vibration data without a knowledge of the individual modal vibration amplitudes. A practical example of the usefulness of this technique is provided by the application of the theoretical calculations to the prediction of the octave band acoustic power output of the picking sticks of an automatic textile loom. Calculations are made of the expected octave band sound pressure levels based on measured acceleration data. These theoretical levels are subsequently compared with actual sound pressure level measurements of loom noise

Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by l,d-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model l,d-transpeptidase (Ldt) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldt. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation

Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type ‘GRASP' domains. Fas/CD95 contains a C-terminal leucine–valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65–Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-XL as a candidate apoptotic binding partner for GRASP65

An OBSL1-Cul7Fbxw8 Ubiquitin Ligase Signaling Mechanism Regulates Golgi Morphology and Dendrite Patterning

The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7Fbxw8 localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7Fbxw8 by independent approaches including Fbxw8 knockdown reveals that Cul7Fbxw8 is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7Fbxw8 also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7Fbxw8 in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7Fbxw8 in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7Fbxw8 ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development

Management of giant pseudomeningoceles after spinal surgery

<p>Abstract</p> <p>Background</p> <p>Pseudomeningoceles are a rare complication after spinal surgery, and studies on these complex formations are few.</p> <p>Methods</p> <p>Between October 2000 and March 2008, 11 patients who developed symptomatic pseudomeningoceles after spinal surgery were recruited. In this retrospective study, we reported our experiences in the management of these complex, symptomatic pseudomeningoceles after spinal surgery. A giant pseudomeningocele was defined as a pseudomeningocele >8 cm in length. We also evaluated the risk factors for the formation of giant pseudomeningoceles.</p> <p>Results</p> <p>All patients were treated successfully with a combined treatment protocol of open revision surgery for extirpation of the pseudomeningoceles, repair of dural tears, and implantation of a subarachnoid catheter for drainage. Surgery-related complications were not observed. Recurrence of pseudomeningocele was not observed for any patient at a mean follow-up of 16.5 months. This result was confirmed by magnetic resonance imaging.</p> <p>Conclusions</p> <p>We conclude that a combined treatment protocol involving open revision surgery for extirpation of pseudomeningoceles, repair of dural tears, and implantation of a subarachnoid catheter for drainage is safe and effective to treat giant pseudomeningoceles.</p

Proteomic Analysis of Rta2p-Dependent Raft-Association of Detergent-Resistant Membranes in Candida albicans

In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans

Ror2 Enhances Polarity and Directional Migration of Primordial Germ Cells

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell

A Genome-Wide RNAi Screen Identifies Regulators of Cholesterol-Modified Hedgehog Secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts