Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner

Lung contains both Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities. Addition of Triton X-100 (0.5%) or chlorpromazine (1 mM) leads to a marked increase in the total phosphatidate phosphohydrolase activity in rat lung microsomes (microsomal fractions), but a decrease in the Mg2+-dependent activity. These observations suggest that the Mg2+-independent activity is stimulated, whereas the Mg2+-dependent activity is inhibited. However, the possibility exists that Triton X-100 could stimulate the Mg2+-dependent enzymic activity in an Mg2+-independent manner. In addition, the positively charged amphiphilic drug could be replacing the enzyme's requirement for Mg2+. These two possibilities were examined by using subcellular fractions in which the Mg2+-dependent phosphatidate phosphohydrolase had been abolished by heat treatment at 55 degrees C for 15 min. Heat treatment does not affect the microsomal Mg2+-independent phosphohydrolase to any great extent. Since the 6-8-fold stimulations due to Triton X-100 and chlorpromazine are retained after heat treatment of this fraction, the Mg2+-independent activity must be involved. Addition of Triton X-100 and chlorpromazine to cytosol virtually abolishes the Mg2+-dependent phosphatidate phosphohydrolase activity and decreases the Mg2+-independent activity by half. Heat treatment also abolishes the Mg2+-dependent activity and decreases the Mg2+-independent activity by over half. The Mg2+-independent phosphatidate phosphohydrolase activity remaining after heat treatment was not affected by Triton X-100 or chlorpromazine. These studies demonstrate that Triton X-100 and chlorpromazine specifically stimulate the heat-stable Mg2+-independent phosphatidate phosphohydrolase activity in rat lung microsomes. In contrast, the heat-labile Mg2+-independent phosphatidate phosphohydrolase activities in cytosol are inhibited by these reagents. Triton X-100 and chlorpromazine inhibit the Mg2+-dependent phosphatidate phosphohydrolase activities in both rat lung microsomes and cytosol. These results are consistent with the view that a single Mg2+-dependent phosphatidate phosphohydrolase present in both microsomes and cytosol is specifically involved in glycerolipid metabolism.

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Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner

Infection and Immunity ◽

10.1128/iai.00534-07 ◽

2007 ◽

Vol 75 (10) ◽

pp. 4719-4727 ◽

Cited By ~ 24

Author(s):

Dhammika N. Atapattu ◽

Charles J. Czuprynski

Keyword(s):

Lipid Rafts ◽

Actin Polymerization ◽

Bacterial Pathogen ◽

Dominant Negative ◽

Mannheimia Haemolytica ◽

Complex Nature ◽

Dependent Manner ◽

Coated Pits ◽

Independent Manner ◽

Dynamin 2

ABSTRACT Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the β2-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.

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Cwc25 Is a Novel Splicing Factor Required after Prp2 and Yju2 To Facilitate the First Catalytic Reaction

Molecular and Cellular Biology ◽

10.1128/mcb.00773-09 ◽

2009 ◽

Vol 29 (21) ◽

pp. 5671-5678 ◽

Cited By ~ 54

Author(s):

Ying-Fang Chiu ◽

Yen-Chi Liu ◽

Ting-Wei Chiang ◽

Tzu-Chi Yeh ◽

Chi-Kang Tseng ◽

...

Keyword(s):

Heat Treatment ◽

Branch Point ◽

Splice Site ◽

Catalytic Reaction ◽

Mrna Splicing ◽

Splicing Factor ◽

Independent Manner ◽

Heat Resistant ◽

Low Concentrations ◽

Spliceosome Activation

ABSTRACT Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn2+. These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5′ splice site and the branch point during the first catalytic reaction.

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The Precipitation of Si in AlCuSi Thin Films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070618 ◽

1973 ◽

Vol 31 ◽

pp. 34-35 ◽

Cited By ~ 1

Author(s):

R. M. Anderson

Keyword(s):

Electron Microscopy ◽

Thin Films ◽

Heat Treatment ◽

Solid Solution ◽

Integrated Circuit ◽

Copper Film ◽

Solution Concentration ◽

X Ray ◽

Silicon Thin Films ◽

Before And After

Aluminum-copper-silicon thin films have been considered as an interconnection metallurgy for integrated circuit applications. Various schemes have been proposed to incorporate small percent-ages of silicon into films that typically contain two to five percent copper. We undertook a study of the total effect of silicon on the aluminum copper film as revealed by transmission electron microscopy, scanning electron microscopy, x-ray diffraction and ion microprobe techniques as a function of the various deposition methods.X-ray investigations noted a change in solid solution concentration as a function of Si content before and after heat-treatment. The amount of solid solution in the Al increased with heat-treatment for films with ≥2% silicon and decreased for films <2% silicon.

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The influence of heat treatment on the fiber/matrix interface in a SiC-reinforced glassceramic

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100105771 ◽

1988 ◽

Vol 46 ◽

pp. 742-743

Author(s):

E. Bischoff ◽

O. Sbaizero

Keyword(s):

Heat Treatment ◽

Aluminum Silicate ◽

Lithium Aluminum ◽

Interfacial Region ◽

Specimen Preparation ◽

Crack Wake ◽

Pull Out ◽

Annealed Material ◽

Ultrasonic Drilling ◽

Fiber Matrix Interface

Fiber or whisker reinforced ceramics show improved toughness and strength. Bridging by intact fibers in the crack wake and fiber pull-out after failure contribute to the additional toughness. These processes are strongly influenced by the sliding and debonding resistance of the interfacial region. The present study examines the interface in a laminated 0/90 composite consisting of SiC (Nicalon) fibers in a lithium-aluminum-silicate (LAS) glass-ceramic matrix. The material shows systematic changes in sliding resistance upon heat treatment.As-processed samples were annealed in air at 800 °C for 2, 4, 8, 16 and 100 h, and for comparison, in helium at 800 °C for 4 h. TEM specimen preparation of as processed and annealed material was performed with special care by cutting along directions having the fibers normal and parallel to the section plane, ultrasonic drilling, dimpling to 100 pm and final ionthinning. The specimen were lightly coated with Carbon and examined in an analytical TEM operated at 200 kV.

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Strain-induced martensite effects on transgranular carbide precipitation in type 304 stainless steels

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087331 ◽

1991 ◽

Vol 49 ◽

pp. 604-605

Author(s):

A.H. Advani ◽

L.E. Murr ◽

D. Matlock

Keyword(s):

Heat Treatment ◽

Grain Boundary ◽

Stress Corrosion Cracking ◽

Stainless Steels ◽

Deformation Twin ◽

Austenitic Stainless Steels ◽

Carbide Precipitation ◽

Strain Induced Martensite ◽

Type 304

Thermomechanically induced strain is a key variable producing accelerated carbide precipitation, sensitization and stress corrosion cracking in austenitic stainless steels (SS). Recent work has indicated that higher levels of strain (above 20%) also produce transgranular (TG) carbide precipitation and corrosion simultaneous with the grain boundary phenomenon in 316 SS. Transgranular precipitates were noted to form primarily on deformation twin-fault planes and their intersections in 316 SS.Briant has indicated that TG precipitation in 316 SS is significantly different from 304 SS due to the formation of strain-induced martensite on 304 SS, though an understanding of the role of martensite on the process has not been developed. This study is concerned with evaluating the effects of strain and strain-induced martensite on TG carbide precipitation in 304 SS. The study was performed on samples of a 0.051%C-304 SS deformed to 33% followed by heat treatment at 670°C for 1 h.

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Effect of a Complex Heat Treatment on the Structure of 300M Steel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098071 ◽

1981 ◽

Vol 39 ◽

pp. 312-313

Author(s):

R. Padmanabhan ◽

W. E. Wood

Keyword(s):

Heat Treatment ◽

Metal Carbides ◽

Austenite Grain Size ◽

300M Steel ◽

Heat Treated ◽

Strain Fracture ◽

Prior Austenite Grain Size ◽

Short Time ◽

Final Tempering ◽

Similar Yield

Intermediate high temperature tempering prior to subsequent reaustenitization has been shown to double the plane strain fracture toughness as compared to conventionally heat treated UHSLA steels, at similar yield strength levels. The precipitation (during tempering) of metal carbides and their subsequent partial redissolution and refinement (during reaustenitization), in addition to the reduction in the prior austenite grain size during the cycling operation have all been suggested to contribute to the observed improvement in the mechanical properties. In this investigation, 300M steel was initially austenitized at 1143°K and then subjected to intermediate tempering at 923°K for 1 hr. before reaustenitizing at 1123°K for a short time and final tempering at 583°K. The changes in the microstructure responsible for the improvement in the properties have been studied and compared with conventionally heat treated steel. Fig. 1 shows interlath films of retained austenite produced during conventionally heat treatment.

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Crystallization of MgO • Al2 • SiO2 • ZrO2 glasses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100143778 ◽

1986 ◽

Vol 44 ◽

pp. 440-441

Author(s):

M. A. McCoy

Keyword(s):

Mechanical Properties ◽

Heat Treatment ◽

Powder Processing ◽

Glass Composition ◽

Ceramic Powder ◽

Transformation Toughening ◽

Ceramic Powder Processing ◽

Processing Techniques ◽

Ceramic Matrices

Transformation toughening by ZrO2 inclusions in various ceramic matrices has led to improved mechanical properties in these materials. Although the processing of these materials usually involves standard ceramic powder processing techniques, an alternate method of producing ZrO2 particles involves the devtrification of a ZrO2-containing glass. In this study the effects of glass composition (ZrO2 concentration) and heat treatment on the morphology of the crystallization products in a MgO•Al2•SiO2•ZrO2 glass was investigated.

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Microstructural Development in Nb-Ti Multifilamentary Superconductors During Processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100117911 ◽

1985 ◽

Vol 43 ◽

pp. 190-191

Author(s):

A. W. West

Keyword(s):

Heat Treatment ◽

Critical Current Density ◽

Copper Matrix ◽

Wire Drawing ◽

Heat Treatments ◽

Microstructural Development ◽

Final Size ◽

Density Values ◽

The Wire ◽

Multifilamentary Superconductors

The influence of the filament microstructure on the critical current density values, Jc, of Nb-Ti multifilamentary superconducting composites has been well documented. However the development of these microstructures during composite processing is still under investigation.During manufacture, the multifilamentary composite is given several heat treatments interspersed in the wire-drawing schedule. Typically, these heat treatments are for 5 to 80 hours at temperatures between 523 and 573K. A short heat treatment of approximately 3 hours at 573K is usually given to the wire at final size. Originally this heat treatment was given to soften the copper matrix, but recent work has shown that it can markedly change both the Jc value and microstructure of the composite.

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High-Temperature Instability of A Cr2Nb-Nb(Cr) Microlaminate Composite

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100164337 ◽

1996 ◽

Vol 54 ◽

pp. 374-375

Author(s):

M. Larsen ◽

R.G. Rowe ◽

D.W. Skelly

Keyword(s):

Heat Treatment ◽

Solid Solution ◽

High Temperature ◽

Elevated Temperatures ◽

Aircraft Engine ◽

Lattice Parameter ◽

Microstructural Stability ◽

Elevated Temperature Strength ◽

Cross Sectional ◽

Bcc Structure

Microlaminate composites consisting of alternating layers of a high temperature intermetallic compound for elevated temperature strength and a ductile refractory metal for toughening may have uses in aircraft engine turbines. Microstructural stability at elevated temperatures is a crucial requirement for these composites. A microlaminate composite consisting of alternating layers of Cr2Nb and Nb(Cr) was produced by vapor phase deposition. The stability of the layers at elevated temperatures was investigated by cross-sectional TEM.The as-deposited composite consists of layers of a Nb(Cr) solid solution with a composition in atomic percent of 91% Nb and 9% Cr. It has a bcc structure with highly elongated grains. Alternating with this Nb(Cr) layer is the Cr2Nb layer. However, this layer has deposited as a fine grain Cr(Nb) solid solution with a metastable bcc structure and a lattice parameter about half way between that of pure Nb and pure Cr. The atomic composition of this layer is 60% Cr and 40% Nb. The interface between the layers in the as-deposited condition appears very flat (figure 1). After a two hour, 1200 °C heat treatment, the metastable Cr(Nb) layer transforms to the Cr2Nb phase with the C15 cubic structure. Grain coarsening occurs in the Nb(Cr) layer and the interface between the layers roughen. The roughening of the interface is a prelude to an instability of the interface at higher heat treatment temperatures with perturbations of the Cr2Nb grains penetrating into the Nb(Cr) layer.

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