Microtensile measurements of single trabeculae stiffness in human femur

In this paper, the authors perform microtensile tests of single trabeculae excised from a human femur head. One of the main issues of this work is to establish some experimental procedures for preparing and testing the specimens. The use of a well-characterized microtensile apparatus allows for a low intraspecimen dispersion of the measured stiffness. Tensile/compressive tests were chosen because they appear less sensitive to errors in the cross-sectional area measurements with respect to bending tests. By these considerations, some tensile/compressive tests of plate-like trabecular specimens have been carried out. Typical stiffness values are 74.2+/-0.7Nmm(-1) for tensile tests, and 58.9+/-0.6Nmm(-1) for compressive test. Another compressive test performed on a shorter specimen yielded a stiffness value of 148.3+/-5.3Nmm(-1). The maximum applied load was about 0.5N. Rough measurements of specimens sizes yielded a Young's modulus value ranging from 1.41 to 1.89GPa.     

Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells

A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [35S]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.     

Comparison of mechanical properties of orthodontic nickel-titanium wires]

Two packages each, containing 10 wires per package, of different batches of 25 different types of orthodontic archwires made of super-elastic nickel-titanium alloys measuring 0.41 x 0.56 mm2, were investigated. The wires were characterized by obtaining the following measurements at an ambient temperature of 37 degrees: a three-point bending test with the supporting points spaced 10 mm apart, and determination of the torque/bending angle curves using a pure bending test. The force/deflection curves provided the parameters characterizing the super-elastic unloading plateau: average force, slope and endpoint. From the torque/bending angle curves, the parameters average torque, plateau endpoint and the elasticity parameters were determined. Average force (0.8-4.5 N), endpoint (0.2-0.9 mm) and the slope of the unloading plateau (0.2-2.1 N/mm) of the three-point bending test clearly differed for individual wires. Significant differences were also seen for average torque (1.5-11.5 Nmm), unloading plateau endpoint (2.7-20.0 degrees) and elasticity parameters epsilon 4, E4, E5 and E6 in the pure bending test. Individual batches showed only minor differences. The results permit the conclusion to be drawn that super-elasticity is applicable to only a small portion of the wires examined. Although other wires showed super-elastic behaviour, the unloading plateaus has a force level of up to 6 N, and cannot be recommended for orthodontic application. The super-elastic plateau is often of use only for deflections greater than 1.5 mm. The use of super-elastic archwires made of nickel-titanium alloys makes sense only when the elastic properties of the respective wires are known. This makes the provision by the manufacturer of relevant data on the elastic properties of wires a necessity.     

Cellular localization and antigenic characterization of crimean-congo hemorrhagic fever virus glycoproteins

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G(N) and G(C). To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G(N) and G(C). Using these MAbs, we found that G(N) predominantly colocalized with a Golgi marker when expressed alone or with G(C), while G(C) was transported to the Golgi apparatus only in the presence of G(N). Both proteins remained endo-beta-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the G(N) ectodomain, because a soluble version of G(N) lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G(N) and G(C) also resulted in localization of soluble G(C) to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G(N) precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G(N) when it was expressed alone but were dispensable when G(C) was coexpressed. In neutralization assays on SW-13 cells, MAbs to G(C), but not to G(N), prevented CCHFV infection. However, only a subset of G(C) MAbs protected mice in passive-immunization experiments, while some nonneutralizing G(N) MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G(C).     

Histochemical study of TPPase and NDPase in the human nervous tissue,with particular reference to glia cells

Summary TPPase and IDPase activities were investigated in normal and reactive human nervous tissue with three divalent ions as activators at different concentrations. Enzyme distribution in neuronal Golgi apparatus, vessels and glia cells is described. In glia cells IDP was hydrolyzed almost equally with the different ions at the concentration of 0.005 M, while TPP was only weakly hydrolyzed with Ca⁺⁺ and Mg⁺⁺. Small positive structures in the cells remained positive and for these a correspondence with the Golgi apparatus is suggested.Partially supported by Consiglio Nazionale delle Ricerche (C.N.R), Rome.     

Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein

As Rift Valley fever (RVF) virus, and probably all members of the family Bunyaviridae, matures in the Golgi apparatus, the targeting of the virus glycoproteins to the Golgi apparatus plays a pivotal role in the virus replication cycle. No consensus Golgi localization motif appears to be shared among the glycoproteins of these viruses. The viruses of the family Bunyaviridae synthesize their glycoproteins, G(N) and G(C), as a polyprotein. The Golgi localization signal of RVF virus has been shown to reside within the G(N) protein by use of a plasmid-based transient expression system to synthesize individual G(N) and G(C) proteins. While the distribution of individually expressed G(N) significantly overlaps with cellular Golgi proteins such as beta-COP and GS-28, G(C) expressed in the absence of G(N) localizes to the endoplasmic reticulum. Further analysis of expressed G(N) truncated proteins and green fluorescent protein/G(N) chimeric proteins demonstrated that the RVF virus Golgi localization signal mapped to a 48-amino-acid region of G(N) encompassing the 20-amino-acid transmembrane domain and the adjacent 28 amino acids of the cytosolic tail.     

Design,Analysis and Experimental Performance of a Bionic Piezoelectric Rotary Actuator

This study presents a piezoelectric rotary actuator which is equipped with a bionic driving mechanism imitating the centipede foot.The configuration and the operational principle are introduced in detail.The movement model is established to analyze the motion of the actuator.We establish a set of experimental system and corresponding experiments are conducted to evaluate the characteristics of the prototype.The results indicate that the prototype can be operated stably step by step and all steps have high reproducibility.The driving resolutions in forward and backward motions are 2.31 μrad and 1.83 μrad,respectively.The prototype can also output a relatively accurate circular motion and the maximum output torques in forward and backward directions are 76.4 Nmm and 70.6 Nmm,respectively.Under driving frequency of 1 Hz,the maximum angular velocities in forward and backward directions are 1029.3 μrad·s-1 and 1165 μrad·s-1 when the driving voltage is 120 V.Under driving voltage of 60 V,the angular velocities in forward and backward motions can be up to 235100 μtrad·s-1 and 153650 prad·s-1 when the driving frequency is 1024 Hz.We can obtain the satisfactory angular velocity by choosing a proper driving voltage and frequency for the actuator.     

A strategy for discovering biologically active compounds with high probability in traditional Chinese herb remedies: an application of saiboku-to in bronchial asthma.

A novel strategy for discovering biologically active components in traditional Chinese herb remedies was performed from a pharmacokinetic view. The hypothesis was that the active compounds should appear in blood and urine with appropriate blood concentrations and urinary excretion rates after the administration of herbal-extract mixtures. In this research, we applied our procedures to Saiboku-To, one of the most popular Chinese herbal medicines in Japan. Consisting of 10 different plant extracts, it is used for the treatment of bronchial asthma. The analytical method adopted was a rapid-flow fractionation (RFF) for extraction-fractionation of lipophilic components in urine followed by silica-gel high-performance liquid chromatography (HPLC) equipped with a multichannel ultraviolet (uv) absorption detector. beta-D-Glucuronidase-treated urine samples collected before and after the administration of Saiboku-To to healthy and asthmatic subjects were treated with the RFF apparatus to afford three pH-dependent fractions: strongly acidic (S), weakly acidic (W), and neutral (N). HPLC of these fractions, monitored by the multichannel uv detector, showed three new peaks in the postadministrative urine: one in the N fraction, two in the W fraction, and none in the S fraction. A compound in the N fraction was identified with authentic magnolol, a major component in Magnolia officinalis. Two compounds in the W fraction were identified by comparison with authentic samples as 8,9-dihydroxydihydromagnolol and liquiritigenin, metabolites previously isolated from M. officinalis and Glycyrrhiza glabra, respectively.     

Direct measurement of nitrogen fixation by Trifolium repens L. and Alnus glutinosa L. using 15N2

A closed-system flow-through enclosure apparatus was used tomeasure symbiotic nitrogen fixation directly. A legume-basedsystem comprising 6-week-old Trifolium repens L. (white clovercv. Blanca) growing with Lolium perenne L. (perennial ryegrasscv. Trani) in an agricultural soil was incubated for 19 d ina ¹⁵N-enriched atmosphere (mean value 3.663 atom%). An actinorhizal-basedsystem comprising 1 -year-old Alnus glutinosa L. (alder) saplingsgrowing with Festuca rubra L. (red fescue) in open-cast coalspoil was incubated for 21 d in a ¹⁵N-enriched atmosphere (meanvalue 3.265 atom%). Indirect estimates of N2 fixation were carriedout concurrently using N difference and ¹⁵N isotope dilutiontechniques. The theory underlying the three techniques and modificationswhich were adopted for comparative purposes are discussed. Thedirect measurements of N2 fixation were then compared with theindirect estimates using P_inc, the proportion of the N incrementduring the measurement period that was derived from fixation.The simple N difference method gave similar values for Pinc(0.94 and 0.97) as those derived from more complicated isotopemethodologies, both indirect (0.91) and direct (0.90). Valuesfor alder were far more variable, ranging from 0.16 to 0.92;this was due largely to variability within the trees and a verysmall N increment during the measurement period. Key words: N₂ fixation, ¹⁵N₂, white clover, alder, enclosure apparatus     

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion- protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta- galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.      

Structure of the intracellular part of the motility apparatus of halobacteria

he electron microscopic study of the structure of the motility apparatus of the archaea Halobacterium salinarium 4W12 and Natronobacterium magadii confirmed our earlier observation that the motility apparatus of halobacteria contains an intracellular disk-shaped lamellar structure (DLS). Polar cap structures (PCSs) isolated from the halobacterium were preliminarily identified as the DLSs. The PCSs in complexes with flagella were also isolated from the haloalkaliphilic bacterium N. magadii. The specific structure of the archaeal motility apparatus is discussed.     

Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar membrane and a weak intracellular staining was demonstrated. No staining was detected in the basolateral membrane. Likewise, the immunogold labelling on Epon-embedded sections was concentrated in the microvillar membrane, whereas the basolateral membrane did not contain significant amounts of labelling. Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar membrane by smooth vesicles having a diameter about 70 to 100 nm and does not pass the basolateral membrane on its way to the brush border membrane.     

Toward an MRI-based method to measure non-uniform cartilage deformation: an MRI-cyclic loading apparatus system and steady-state cyclic displacement of articular cartilage under compressive loading

Recent magnetic resonance imaging (MRI) techniques have shown potential for measuring non-uniform deformations throughout the volume (i.e. three-dimensional (3D) deformations) in small orthopedic tissues such as articular cartilage. However, to analyze cartilage deformation using MRI techniques, a system is required which can construct images from multiple acquisitions of MRI signals from the cartilage in both the underformed and deformed states. The objectives of the work reported in this article were to 1) design an apparatus that could apply highly repeatable cyclic compressive loads of 400 N and operate in the bore of an MRI scanner, 2) demonstrate that the apparatus and MRI scanner can be successfully integrated to observe 3D deformations in a phantom material, 3) use the apparatus to determine the load cycle necessary to achieve a steady-state deformation response in normal bovine articular cartilage samples using a flat-surfaced and nonporous indentor in unconfined compression. Composed of electronic and pneumatic components, the apparatus regulated pressure to a double-acting pneumatic cylinder so that (1) load-controlled compression cycles were applied to cartilage samples immersed in a saline bath, (2) loading and recovery periods within a cycle varied in time duration, and (3) load magnitude varied so that the stress applied to cartilage samples was within typical physiological ranges. In addition the apparatus allowed gating for MR image acquisition, and operation within the bore of an MRI scanner without creating image artifacts. The apparatus demonstrated high repeatability in load application with a standard deviation of 1.8% of the mean 400 N load applied. When the apparatus was integrated with an MRI scanner programmed with appropriate pulse sequences, images of a phantom material in both the underformed and deformed states were constructed by assembling data acquired through multiple signal acquisitions. Additionally, the number of cycles to reach a steady-state response in normal bovine articular cartilage was 49 for a total cycle duration of 5 seconds, but decreased to 33 and 27 for increasing total cycle durations of 10 and 15 seconds, respectively. Once the steady-state response was achieved, 95% of all displacements were within +/- 7.42 microns of the mean displacement, indicating that the displacement response to the cyclic loads was highly repeatable. With this performance, the MRI-loading apparatus system meets the requirements to create images of articular cartilage from which 3D deformation can be determined.     

N-linked glycan-dependent interaction of CD63 with CXCR4 at the Golgi apparatus induces downregulation of CXCR4

Efficient downregulation of CXCR4 cell surface expression by introduction of the CD63 gene has previously been reported by us. In the present study, it was found that CD63 and its mutant efficiently interact with CXCR4 in live cells and that CD63-induced downregulation and interaction are significantly abrogated by the N- linked glycosylation inhibitor, TM. Furthermore, the downregulation and interaction were clearly attenuated by alternation of all three N- linked glycosylation sites in CD63. Either CD63 or CD63ΔN formed a complex with CXCR4 at the Golgi apparatus and the late endosomes, while CD63 GD mutants lost the ability to form a complex with CXCR4 exclusively at the Golgi apparatus. These findings suggest that CD63 interacts with CXCR4 through the N- linked glycans-portion of the CD63 protein and that the complex induces direction of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane. At the Golgi apparatus, there may be lysosome protein (CD63)-associated machinery that influences trafficking of other membrane proteins.     

Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa.

Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa. Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features. Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane. To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization. We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.     

狭基巢蕨叶表皮的结构和气孔器发育的观察

狭基巢蕨Ｎｅｏｔｏｐｔｅｒｉｓａｎｔｒｏｐｈｙｏｉｄｅｓ（Ｃｈｒｉｓｔ）Ｃｈｉｎｇ叶片的上表皮无气孔器,仅具表皮细胞,下表皮由表皮细胞和气孔器组成,气孔指数为２．５。上下表皮细胞和气孔器的细胞中均含有叶绿体。每个气孔器由２个肾形的保卫细胞和２～６个副卫细胞组成,其中以３个和４个副卫细胞的占绝大多数（３细胞的占４５．１％,４细胞的占４３．５％）。从发育上看,气孔器原始细胞进行２次分裂,产生２个保卫细胞和１个同源的副卫细胞。气孔器的发育过程大体可分为４个时期：（１）气孔器原始细胞的分化和分裂期;（２）保卫细胞母细胞成熟期;（３）保卫细胞母细胞分裂和气孔器幼期;（４）气孔器成熟期。狭基巢蕨的气孔器属于中周型     

Genetic organization of the hrp gene cluster in Acidovorax avenae strain N1141 and a novel effector protein that elicits immune responses in rice (Oryza sativa L.)

The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.     

Biological function of the low-pH, fusion-inactive conformation of rabies virus glycoprotein (G): G is transported in a fusion-inactive state-like conformation.

The glycoprotein (G) of rabies virus can assume at least three different conformations: the native (N) state detected at the viral surface above pH 7; the activated (A) hydrophobic state, which is probably involved in the first steps of the fusion process; and the fusion-inactive (I) conformation. There is a pH-dependent equilibrium between these states, the equilibrium being shifted towards the I state at low pH. It has been supposed that the transition from the N to the I state mediates membrane fusion. By using a lipid-mixing assay, we studied the kinetics of fusion and fusion inactivation for two rabies virus strains, PV and CVS. In addition, by using electron microscopy and a trypsin sensitivity assay, we analyzed the kinetics of the conformational change towards the I state for both strains. Although the PV strain fuses faster, inactivation and the conformational change of PV G occur more slowly than for the CVS strain. This suggests that the structural transition towards the I state is irrelevant to the fusion process. Immunofluorescence and immunoprecipitation experiments performed with infected cells and two different monoclonal antibodies, one specific for the N form of G and one which recognizes both the N and the I states, suggest that G is transported in an I state-like conformation through the Golgi apparatus and acquires its N structure only near or at the cell surface. We propose that the role of the I state is to avoid unspecific fusion during transport of G in the acidic Golgi vesicles.     

Social diffusion of novel foraging methods in brown capuchin monkeys (Cebus apella)

It has been reported that wild capuchin monkeys exhibit several group-specific behavioural traditions. By contrast, experiments have found little evidence for the social learning assumed necessary to support such traditions. The present study used a diffusion chain paradigm to investigate whether a novel foraging task could be observationally learned by capuchins (Cebus apella) and then transmitted along a chain of individuals. We used a two-action paradigm to control for independent learning. Either of two methods (lift or slide) could be used to open the door of a foraging apparatus to retrieve food. Two chains were tested (N1=4; N2=5), each beginning with an experimenter-trained model who demonstrated to a partner its group-specific method for opening the foraging apparatus. After the demonstration, if the observer was able to open the apparatus 20 times by either method, then it became the demonstrator for a new subject, thus simulating the spread of a foraging tradition among 'generations' of group members. Each method was transmitted along these respective chains with high fidelity, echoing similar results presently available only for chimpanzees and children. These results provide the first clear evidence for faithful diffusion of alternative foraging methods in monkeys, consistent with claims for capuchin traditions in the wild.