Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

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Electrophoretic and electron microscopic examination of the adductor and diductor muscles of an articulate brachiopod, Terebratalia transversa

Canadian Journal of Zoology ◽

10.1139/z82-082 ◽

1982 ◽

Vol 60 (4) ◽

pp. 550-559 ◽

Cited By ~ 4

Author(s):

William P. Eshleman ◽

Jerrel L. Wilkens ◽

Michael J. Cavey

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Electron Microscopic Examination ◽

Light Chains ◽

Electron Microscopic ◽

Electrophoretic Patterns ◽

Transmission Electron ◽

Adductor Muscles ◽

Regulation Of Contraction

The proteins of the striated adductor muscles, smooth adductor muscles, and diductor muscles of the articulate brachiopod Terebratalia transversa have been examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Electrophoretic patterns indicate the presence of paramyosin in all of these valve muscles. Tentative identification has also been made of the proteins responsible for actin and for myosin regulation of contraction (troponin–tropomyosin and myosin light chains, respectively). The myofilaments of the striated adductor cells, smooth adductor cells, and diductor cells have been characterized by transmission electron microscopy. The smooth adductor cells and the diductor cells exhibit very thick myofilaments which are fusiform in shape, exceptionally long, and axially banded. Morphological features of these thick myofilaments are consistent with those of paramyosin filaments found in other muscles and myoepithelia. Although the striated adductor cells contain paramyosin, it is not manifest in the thick myofilaments.

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Characterization of the yrbA Gene ofBacillus subtilis, Involved in Resistance and Germination of Spores

Journal of Bacteriology ◽

10.1128/jb.181.16.4986-4994.1999 ◽

1999 ◽

Vol 181 (16) ◽

pp. 4986-4994 ◽

Cited By ~ 52

Author(s):

Hiromu Takamatsu ◽

Takeko Kodama ◽

Tatsuo Nakayama ◽

Kazuhito Watabe

Keyword(s):

Optical Density ◽

Protein Profile ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Electron Microscopic Examination ◽

Coat Proteins ◽

Electron Microscopic ◽

Consensus Sequences ◽

Insertional Inactivation ◽

Coat Layer

ABSTRACT Insertional inactivation of the yrbA gene ofBacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in varioussig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination ofyrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.

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Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum)

Biochemical Journal ◽

10.1042/bj1900551 ◽

1980 ◽

Vol 190 (3) ◽

pp. 551-561 ◽

Cited By ~ 68

Author(s):

C R Slack ◽

W S Bertaud ◽

B D Shaw ◽

R Holland ◽

J Browse ◽

...

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Electron Microscopic Examination ◽

Carthamus Tinctorius ◽

Oil Body ◽

Oil Bodies ◽

Electron Microscopic ◽

Intact Cells ◽

Glycerolipid Composition ◽

Induced Aggregation

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.

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Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072733 ◽

1973 ◽

Vol 31 ◽

pp. 458-459

Author(s):

K. S. McCarty ◽

R. F. Weave ◽

L. Kemper ◽

F. S. Vogel

Keyword(s):

Mitochondrial Dna ◽

Ethidium Bromide ◽

Microscopic Examination ◽

Agaricus Bisporus ◽

Osmotic Shock ◽

Electron Microscopic Examination ◽

Electron Microscopic ◽

Isolated Mitochondria ◽

Dna Strands ◽

Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

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Artifact-free electron microscopic immunocytochemistry on rat brain tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085666 ◽

1991 ◽

Vol 49 ◽

pp. 272-273

Author(s):

Veronika Burmeister ◽

N. Ludvig ◽

P.C. Jobe

Keyword(s):

Microscopic Examination ◽

Free Electron ◽

Electron Microscopic Examination ◽

Ultrastructural Localization ◽

Rabbit Serum ◽

Electron Microscopic ◽

Electron Microscopic Immunocytochemistry ◽

Phosphate Buffered Saline ◽

Rat Brain Tissue ◽

Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

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Ultrastructural studies of Chlamydia psittaci 6BC variant strains. I. Ultrastructure of the surface layers of egg-passaged 6BC strain

Canadian Journal of Microbiology ◽

10.1139/m73-142 ◽

1973 ◽

Vol 19 (8) ◽

pp. 887-894

Author(s):

Linda Poffenroth ◽

J. W. Costerton ◽

Nonna Kordová ◽

John C. Wilt

Keyword(s):

Chick Embryo ◽

Microscopic Examination ◽

Electron Microscopic Examination ◽

Chlamydia Psittaci ◽

Gram Negative Bacteria ◽

Surface Layers ◽

Electron Microscopic ◽

Gram Negative ◽

Ultrastructural Studies ◽

First Time

Electron microscopic examination of a semipurified Chlamydia psittaci 6BC strain attenuated in chick embryo yolk sac revealed for the first time two morphologically distinct small elementary bodies which differ both in the ultrastructure of their surface layers and in their buoyant densities in sucrose gradients. Also, the morphology of the surface layers of the larger reticulate forms in cell-free systems is described for the first time. Many points of difference between the surface envelopes and internal structure of chlamydial particles and those of Gram-negative bacteria are discussed.

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