Serological and Physicochemical Reactivity of Bovine Erythrocytes before and after Trypsin Treatment

We looked for the presence of prorenin in erythrocytes from normal subjects (n = 8), hypertensive patients (n = 8), and pregnant women (n = 8). Angiotensin I generation was measured at 37 °C, pH 5.7, in the presence of homologous substrate (1400 ng/mL) before and after trypsin activation (100 μg/mL) in (A) haemolyzed erythrocytes, (B) supernatants of haemolyzed erythrocytes, and (C) in the sixth washing of erythrocytes diluted 1:1 with a 0.1 M Tris buffer containing 0.5% bovine serum albumin and protease inhibitors. Haemolyzed erythrocytes generated angiotensin I only after trypsin treatment, and the rate of generation was the same (A) before and (B) after centrifugation at 20000g, indicating the absence of prorenin bound to the cell membranes. When aliquots of the last washing of erythrocytes (C) were tested for angiotensin I generation before and after trypsin, they did not generate angiotensin I, indicating that residual prorenin from the plasma was no longer present in our preparation. Angiotensin I generation by trypsin-treated A and B was completely abolished by preincubation with anti-renin serum. The level of prorenin was not significantly different in the erythrocytes from normal, hypertensive, and pregnant subjects (68 ± 10, 58 ± 7 and 107 ± 17 pg angiotensin I∙mL−1∙h−1, ns) in spite of their very different plasma levels (21 ± 2.5, 17 ± 2.4 and 110 ± 12 ng angiotensin I∙mL−1∙h−1, p < 0.01 for pregnant women compared with both normal and hypertensive subjects). Our data show that prorenin is present in human erythrocytes in fairly constant and clearly detectable amounts, thus suggesting a possible intracellular role for it.Key words: inactive renin, intracellular prorenin, erythrocytes, prorenin.

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Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Journal of Virology ◽

10.1128/jvi.75.13.6052-6061.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 6052-6061 ◽

Cited By ~ 95

Author(s):

Sue E. Crawford ◽

Sharmila K. Mukherjee ◽

Mary K. Estes ◽

Jeffery A. Lawton ◽

Andrea L. Shaw ◽

...

Keyword(s):

Biochemical Analysis ◽

De Novo ◽

Protein Composition ◽

Structural Basis ◽

Trypsin Treatment ◽

Animal Group ◽

Virus Particles ◽

Before And After ◽

Group A Rotaviruses ◽

Group A

ABSTRACT Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5∗ and VP8∗ in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8∗ portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5∗, at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.

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Chymotrypsin and neuraminidase treatment inhibits host cell invasion by Babesia divergens (Phylum Apicomplexa)

Parasitology ◽

10.1017/s0031182002001798 ◽

2002 ◽

Vol 125 (1) ◽

pp. 45-50 ◽

Cited By ~ 14

Author(s):

A. ZINTL ◽

C. WESTBROOK ◽

H. E. SKERRETT ◽

J. S. GRAY ◽

G. MULCAHY

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Inhibitory Effect ◽

Host Cell Invasion ◽

Trypsin Treatment ◽

Parasite Invasion ◽

Neuraminidase Treatment ◽

Babesia Divergens ◽

Erythrocyte Surface ◽

Bovine Erythrocytes

The process of host cell invasion by Babesia divergens is poorly understood and improved knowledge of the mechanism involved could lead to development of measures effective in disease prevention. To investigate parasite ligands on the erythrocyte surface, B. divergens cultures in bovine erythrocytes were transferred into enzyme-treated bovine, human, ovine and equine erythrocytes. Parasite invasion of bovine erythrocytes was not affected by trypsin treatment while treatment with alpha-chymotrypsin led to a reduction in parasite growth of 20–40%. Treatment of bovine and non-bovine erythrocytes with neuraminidase decreased their susceptibility to invasion by up to 97% implicating sialic acid as an important erythrocyte ligand for babesia, but the addition of either bovine or human N-acetylneuraminyl-lactose to B. divergens cultures in bovine erythrocytes had no inhibitory effect.

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Deformation Replication

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068394 ◽

1970 ◽

Vol 28 ◽

pp. 280-281

Author(s):

J. Temple Black

Keyword(s):

Cutting Speed ◽

Machining Process ◽

Depth Of Cut ◽

Rake Face ◽

Orthogonal Machining ◽

Aluminum Chips ◽

Before And After ◽

Materials Used ◽

Glass Knife ◽

Very High

Tool materials used in ultramicrotomy are glass, developed by Latta and Hartmann (1) and diamond, introduced by Fernandez-Moran (2). While diamonds produce more good sections per knife edge than glass, they are expensive; require careful mounting and handling; and are time consuming to clean before and after usage, purchase from vendors (3-6 months waiting time), and regrind. Glass offers an easily accessible, inexpensive material ($0.04 per knife) with very high compressive strength (3) that can be employed in microtomy of metals (4) as well as biological materials. When the orthogonal machining process is being studied, glass offers additional advantages. Sections of metal or plastic can be dried down on the rake face, coated with Au-Pd, and examined directly in the SEM with no additional handling (5). Figure 1 shows aluminum chips microtomed with a 75° glass knife at a cutting speed of 1 mm/sec with a depth of cut of 1000 Å lying on the rake face of the knife.

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The Clinical, Roentgenological and Morphological Effects of an Acute Exposure of Phosgene on the Lungs of Dogs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065237 ◽

1971 ◽

Vol 29 ◽

pp. 236-237

Author(s):

R. F. Bils ◽

W. F. Diller ◽

F. Huth

Keyword(s):

Latent Period ◽

Chemical Industry ◽

Toxic Substance ◽

Lung Edema ◽

X Rays ◽

Beagle Dogs ◽

Male And Female ◽

Morphological Alterations ◽

Before And After ◽

Electron Microscopes

Phosgene still plays an important role as a toxic substance in the chemical industry. Thiess (1968) recently reported observations on numerous cases of phosgene poisoning. A serious difficulty in the clinical handling of phosgene poisoning cases is a relatively long latent period, up to 12 hours, with no obvious signs of severity. At about 12 hours heavy lung edema appears suddenly, however changes can be seen in routine X-rays taken after only a few hours' exposure (Diller et al., 1969). This study was undertaken to correlate these early changes seen by the roengenologist with morphological alterations in the lungs seen in the'light and electron microscopes.Forty-two adult male and female Beagle dogs were selected for these exposure experiments. Treated animals were exposed to 94.5-107-5 ppm phosgene for 10 min. in a 15 m3 chamber. Roentgenograms were made of the thorax of each animal before and after exposure, up to 24 hrs.

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Ultrastructure of Melanin Formation in Curvularia sp., Alternaria sp., and Drechslera Sorokiniana

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079449 ◽

1977 ◽

Vol 35 ◽

pp. 398-399

Author(s):

M. H. Wheeler ◽

W. J. Tolmsoff ◽

A. A. Bell

Keyword(s):

Potassium Phosphate ◽

Thielaviopsis Basicola ◽

Wild Type ◽

Potassium Phosphate Buffer ◽

Transmission Microscopy ◽

Electron Transmission ◽

Melanin Formation ◽

Before And After ◽

Alternaria Sp ◽

Electron Transmission Microscopy

(+)-Scytalone [3,4-dihydro-3,6,8-trihydroxy-l-(2Hj-naphthalenone] and 1,8-di- hydroxynaphthalene (DHN) have been proposed as intermediates of melanin synthesis in the fungi Verticillium dahliae (1, 2, 3, 4) and Thielaviopsis basicola (4, 5). Scytalone is enzymatically dehydrated by V. dahliae to 1,3,8-trihydroxynaphthalene which is then reduced to (-)-vermelone [(-)-3,4- dihydro-3,8-dihydroxy-1(2H)-naphthalenone]. Vermelone is subsequently dehydrated to DHN which is enzymatically polymerized to melanin.Melanin formation in Curvularia sp., Alternaria sp., and Drechslera soro- kiniana was examined by light and electron-transmission microscopy. Wild-type isolates of each fungus were compared with albino mutants before and after treatment with 1 mM scytalone or 0.1 mM DHN in 50 mM potassium phosphate buffer, pH 7.0. Both chemicals were converted to dark pigments in the walls of hyphae and conidia of the albino mutants. The darkened cells were similar in appearance to corresponding cells of the wild types under the light microscope.

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Evaluation of Freezing Methods by Low Temperature X-Ray Diffraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079176 ◽

1977 ◽

Vol 35 ◽

pp. 330-333

Author(s):

T. Gulik-Krzywicki ◽

M.J. Costello

Keyword(s):

Biological Materials ◽

Molecular Organization ◽

X Ray Diffraction ◽

Glass Capillary ◽

X Ray ◽

Rate Dependent ◽

Before And After ◽

Freeze Etching ◽

Diffraction Patterns ◽

Set Up

Freeze-etching electron microscopy is currently one of the best methods for studying molecular organization of biological materials. Its application, however, is still limited by our imprecise knowledge about the perturbations of the original organization which may occur during quenching and fracturing of the samples and during the replication of fractured surfaces. Although it is well known that the preservation of the molecular organization of biological materials is critically dependent on the rate of freezing of the samples, little information is presently available concerning the nature and the extent of freezing-rate dependent perturbations of the original organizations. In order to obtain this information, we have developed a method based on the comparison of x-ray diffraction patterns of samples before and after freezing, prior to fracturing and replication.Our experimental set-up is shown in Fig. 1. The sample to be quenched is placed on its holder which is then mounted on a small metal holder (O) fixed on a glass capillary (p), whose position is controlled by a micromanipulator.

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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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An Electron Microscope Study of Interdiffusion and Compound Formation in Ta-Au Thin Films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070606 ◽

1973 ◽

Vol 31 ◽

pp. 32-33 ◽

Cited By ~ 1

Author(s):

T. C. Tisone ◽

S. Lau

Keyword(s):

Electron Microscope ◽

Electron Microscope Study ◽

Thermally Grown Oxide ◽

Ion Milling ◽

Compound Formation ◽

Technology Application ◽

Transmission Electron ◽

Before And After ◽

Au Thin Films

In a study of the properties of a Ta-Au metallization system for thin film technology application, the interdiffusion between Ta(bcc)-Au, βTa-Au and Ta2M-Au films was studied. Considered here is a discussion of the use of the transmission electron microscope(TEM) in the identification of phases formed and characterization of the film microstructures before and after annealing.The films were deposited by sputtering onto silicon wafers with 5000 Å of thermally grown oxide. The film thicknesses were 2000 Å of Ta and 2000 Å of Au. Samples for TEM observation were prepared by ultrasonically cutting 3mm disks from the wafers. The disks were first chemically etched from the silicon side using a HNO3 :HF(19:5) solution followed by ion milling to perforation of the Au side.

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