scholarly journals Isolation by calcium-dependent translation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4817-4823 ◽  
Author(s):  
C Sjolin ◽  
C Dahlgren
Keyword(s):  
Peptide Mapping ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Human Neutrophils ◽  
Calcium Dependent ◽  
Cytosolic Protein ◽  
Specific Granules

Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti- cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.

The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds

1979 ◽  
Vol 57 (3) ◽  
pp. 279-285 ◽  
Author(s):  
Maire E. Percy ◽  
Lebe Chang ◽  
Catherine Demoliou ◽  
Reuben Baumal
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Disulfide Bridge ◽  
Myeloma Protein ◽  
Kinetics Of

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.

scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Abstract Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

scholarly journals Defensin-rich dense granules of human neutrophils

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 757-765 ◽  
Author(s):  
WG Rice ◽  
T Ganz ◽  
JM Jr Kinkade ◽  
ME Selsted ◽  
RI Lehrer ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Human Neutrophils ◽  
Total Acid ◽  
Density Fractions ◽  
Dense Granules ◽  
Blood Neutrophils

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

scholarly journals Identification and Molecular Analysis of Rough-Colony-Specific Outer Membrane Proteins of Actinobacillus actinomycetemcomitans

1999 ◽  
Vol 67 (6) ◽  
pp. 2901-2908 ◽  
Author(s):  
Elaine M. Haase ◽  
Joyce L. Zmuda ◽  
Frank A. Scannapieco
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Actinobacillus Actinomycetemcomitans ◽  
Genome Database ◽  
Gram Negative ◽  
Genes Encoding

ABSTRACT Actinobacillus actinomycetemcomitans, a gram-negative bacterium isolated from the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadherent, smooth phenotype upon in vitro subculture. The rough colony variant produces abundant fimbriae and autoaggregates, while the smooth colony variant is planktonic and produces scant fimbriae. To begin to understand the significance of colony variation in biofilm formation by A. actinomycetemcomitans, outer membrane protein profiles of four isogenic rough and smooth colony variants were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of these proteins was not found to be dependent on growth phase, oxygen tension, or type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the University of Oklahoma A. actinomycetemcomitans genome database. Two contiguous open reading frames (ORFs) encoding proteins having sequence homology with these proteins were identified. The 43-kDa protein (RcpA [rough colony protein A]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony protein B]) appeared to be unique. The genes encoding these proteins have been cloned from A. actinomycetemcomitans 283 and sequenced. A BLASTX (gapped BLAST) search of the surrounding ORFs revealed homology with other fimbria-related proteins. These data suggest that the genes encoding the 43-kDa (rcpA) and 20-kDa (rcpB) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.

Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1683-1692 ◽  
Author(s):  
Beni B. Wolf ◽  
Joshua C. Goldstein ◽  
Henning R. Stennicke ◽  
Helen Beere ◽  
Gustavo P. Amarante-Mendes ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Platelet Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Phosphatidyl Serine ◽  
Human Platelets ◽  
Independent Manner ◽  
Calcium Dependent

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

scholarly journals Evaluation of the Proline-Rich Antigen ofCoccidioides immitis as a Vaccine Candidate in Mice

1998 ◽  
Vol 66 (8) ◽  
pp. 3519-3522 ◽  
Author(s):  
Theo N. Kirkland ◽  
Fred Finley ◽  
Kris I. Orsborn ◽  
John N. Galgiani
Keyword(s):  
T Cells ◽  
Gel Electrophoresis ◽  
Proliferative Response ◽  
Vaccine Candidate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Coccidioides Immitis ◽  
Primary Amino

ABSTRACT We have expressed the proline-rich antigen (PRA) fromCoccidioides immitis in Escherichia coli and evaluated its potential as a vaccine candidate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein (rPRA) revealed two bands, which exhibited virtually identical primary amino acid sequences. T cells from rPRA-immunized BALB/c mice showed a significant in vitro proliferative response to rPRA. A small but statistically significant proliferative response was also induced by rPRA in T cells from mice immunized with whole-cell coccidioidal vaccines. BALB/c mice immunized with rPRA and challenged intraperitoneally with virulent C. immitis had a greatly reduced fungal burden in their lungs and spleens compared to unvaccinated mice. The number of organisms in the lungs was reduced 500-fold, and similar reductions were observed in the spleens of immunized mice. These studies support the continued development of rPRA as a candidate vaccine for prevention of coccidioidomycosis.

scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

scholarly journals Expression of cmg1, an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans, Increases during Sclerotial Parasitism

2001 ◽  
Vol 67 (2) ◽  
pp. 865-871 ◽  
Author(s):  
Gábor Giczey ◽  
Zoltán Kerényi ◽  
László Fülöp ◽  
László Hornok
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Coniothyrium Minitans ◽  
Calculated Molecular Mass ◽  
Secretion Signal

ABSTRACT During sclerotial infection of Sclerotinia sclerotiorumthe mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiaeINVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth ofS. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml−1, respectively. A single copy of thecmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.

scholarly journals Defensin-rich dense granules of human neutrophils

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 757-765 ◽  
Author(s):  
WG Rice ◽  
T Ganz ◽  
JM Jr Kinkade ◽  
ME Selsted ◽  
RI Lehrer ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Human Neutrophils ◽  
Total Acid ◽  
Density Fractions ◽  
Dense Granules ◽  
Blood Neutrophils

Abstract Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1683-1692 ◽  
Author(s):  
Beni B. Wolf ◽  
Joshua C. Goldstein ◽  
Henning R. Stennicke ◽  
Helen Beere ◽  
Gustavo P. Amarante-Mendes ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Platelet Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Phosphatidyl Serine ◽  
Human Platelets ◽  
Independent Manner ◽  
Calcium Dependent

Abstract Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

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