Localization of actin in the salivary glands of the American dog tick, Dermacentor variabilis

1991 ◽  
Vol 49 ◽  
pp. 320-321
Author(s):  
William Lamoreaux ◽  
Naby Sankhon ◽  
Lewis B. Coons
Keyword(s):  
Salivary Glands ◽  
Dermacentor Variabilis ◽  
Ixodid Ticks ◽  
Dot Blot ◽  
Rhodamine Phalloidin ◽  
Type Iii ◽  
Dog Tick ◽  
Dot Blot Analysis

The salivary glands of ixodid ticks are the primary organs of osmoregulation and the source of pathogen transfer from tick to host. Excess fluid is extracted from the blood meal, moved across the gut wall and into the salivary glands, where it is returned to the host. Previously it has been shown in vivo and in vitro that the type III acinus alternately swells as it fills with fluid and then contracts as the acinus empties, and that cytochalasin D prevents contraction of the type III acini.In this investigation, the rhodamine-phalloidin technique was used to localize actin filaments in the salivary glands of fed mated female ticks. Two microfilament inhibitors were used as controls. Samples were also taken for electron microscopy and dot blot analysis.

scholarly journals Interleukin-12 expression in human lymphomas and nonneoplastic lymphoid disorders

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2182-2188 ◽  
Author(s):  
J Schwaller ◽  
A Tobler ◽  
G Niklaus ◽  
N Hurwitz ◽  
I Hennig ◽  
...  
Keyword(s):  
Lymphoid Cells ◽  
Interleukin 12 ◽  
Dot Blot ◽  
Barr Virus ◽  
Epstein Barr ◽  
Hodgkin's Lymphomas ◽  
Dot Blot Analysis

Interleukin-12 (IL-12), a cytokine with in vitro and in vivo immunomodulatory effects, is produced by lymphocytes and stimulated monocytes. Little is known about the production and possible role of IL-12 in human lymphoproliferative disorders. We examined IL-12 expression by immunohistochemistry using antibodies recognizing the p40, p35 subunits, and the p70 heterodimeric IL-12 protein, and by Northern blot in lymph nodes from patients with Hodgkin's disease (HD), non-Hodgkin's lymphomas (NHL), and nonneoplastic lymphoid lesions. In the majority of the HD cases (28 of 34), IL-12 immunoreaction was found in small lymphoid cells cultured around Hodgkin and Reed-Sternberg (H&RS) cells. No IL-12 signal was seen in H&RS cells. Transcripts for IL-12 were found by Northern and dot blot analysis in 13 of 19 (IL-12 p40) and 11 of 19 (IL-12 p35) cases. The HD cases were further examined for the presence of Epstein-Barr virus (EBV) latent membrane protein (LMP-1). All cases with EBV-LMP-1 positivity (22 of 34 cases) also expressed IL- 12. No IL-12 immunoreaction was found in neoplastic cells of 33 cases of various NHLs, which were all LMP-1 negative and showed no EBV-genome sequence, as assessed by polymerase chain reaction (PCR). In 24 nonneoplastic lymphoid lesions, few dispersed IL-12 positive cells were seen in the parafollicular area and in the sinus of the lymph node. The marked presence of IL-12 in the majority of HD cases indicates that IL-12 might play a role in the pathobiology of HD, suggesting that this cytokine is involved in EBV-positive HD.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

1991 ◽  
Vol 114 (4) ◽  
pp. 773-786 ◽  
Author(s):  
P D Kouklis ◽  
T Papamarcaki ◽  
A Merdes ◽  
S D Georgatos
Keyword(s):  
Potential Role ◽  
Type Iii ◽  
Filament Assembly ◽  
Filament Bundles ◽  
Self Association ◽  
Specific Association ◽  
A Site ◽  
Idiotypic Antibody

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

scholarly journals The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

2020 ◽  
Vol 295 (51) ◽  
pp. 17770-17780
Author(s):  
Ikuko Hayashi
Keyword(s):  
Lipid Membranes ◽  
Binding Protein ◽  
Coiled Coil ◽  
Type Iii ◽  
Daughter Cells ◽  
Lysine Residues ◽  
Stable Maintenance ◽  
Biochemical Analyses

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

scholarly journals Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1523
Author(s):  
Isabelle Anna Zink ◽  
Erika Wimmer ◽  
Christa Schleper
Keyword(s):  
Comparative Analysis ◽  
Molecular Mechanisms ◽  
Model Organisms ◽  
Special Focus ◽  
Natural Environments ◽  
Type I ◽  
Accessory Proteins ◽  
Type Iii

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

Murine cytomegalovirus – Pseudomonas synergistic infections: comparison of virulent and attenuated virus

1987 ◽  
Vol 33 (10) ◽  
pp. 923-927 ◽  
Author(s):  
B. Kournikakis ◽  
L. A. Babiuk
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Salivary Glands ◽  
The Body ◽  
Murine Cytomegalovirus ◽  
Viral Neutralization ◽  
Lymphatic Circulation ◽  
Pulmonary Clearance ◽  
Attenuated Virus

The synergistic interactions between Pseudomonas aeruginosa and virulent or attenuated murine cytomegalovirus (MCMV) were compared in vivo. Virulent MCMV challenge at a dose of 5 × 105 pfu/mouse intraperitioneally, followed by intranasal superinfection with 5 × 106 cfu/mouse of Pseudomonas aeruginosa after 48 h resulted in greater than 80% mortality, apparently owing to a failure of pulmonary clearance mechanisms. Single infections, or the use of attenuated MCMV in synergistic infections, did not result in significant morbidity or mortality. Infection with virulent MCMV in vivo resulted in the rapid spread of virus to the lung, liver, and spleen, followed later by spread to the salivary glands. Attenuated virus was detected in salivary glands only. Virulent MCMV was more effective in adsorbing to, or infecting, spleen cells in vitro than attenuated virus. Viral neutralization experiments using anti-viral serum, rabbit complement, and anti-mouse IgG confirmed the presence of a nonneutralizing antibody on the surface of the virulent virus. Our results suggest that the presence of the nonneutralizing antibody on virulent MCMV allows the virus to preferentially infect, or adsorb to, Fc+ cells in the peritoneum. These cells may then carry the virus, via the lymphatic circulation, to other areas of the body, resulting in the replication of virus in multiple organs. Virus replication in the lung may, in part, be the cause of the observed suppression of pulmonary clearance.

scholarly journals Attenuation of late simian virus 40 mRNA synthesis is enhanced by the agnoprotein and is temporally regulated in isolated nuclear systems.

1985 ◽  
Vol 5 (6) ◽  
pp. 1327-1334 ◽  
Author(s):  
N Hay ◽  
Y Aloni
Keyword(s):  
Gel Electrophoresis ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type Virus ◽  
Insertion Mutant ◽  
Dot Blot ◽  
Nuclear Systems

Studies were performed to verify the physiological significance of attenuation in the life cycle of simian virus 40 and the role of agnoprotein in this process. For these purposes, nuclei were isolated at various times after infection and incubated in vitro in the presence of [alpha-32P]UTP under the standard conditions which lead to attenuation. Attenuation was evident by the production of a 94-nucleotide attenuator RNA, revealed by gel electrophoresis. In parallel, the synthesis of agnoprotein was studied at various times after infection by labeling the cells for 3 h with [14C]arginine, lysing them, and analyzing the labeled proteins by gel electrophoresis. Both attenuation and the synthesis of agnoprotein were predominant towards the end of the infectious cycle. At earlier times, there was almost no attenuation and no synthesis of agnoprotein. Moreover, there was almost no attenuation even at the latest times after infection in nuclei isolated from cells infected with simian virus 40 deletion mutants that do not synthesize agnoprotein. Finally, analysis by dot blot hybridization showed higher amounts of cytoplasmic viral RNA in cells infected with an agnoprotein gene insertion mutant, delta 79, that does not produce agnoprotein, compared with cells infected with wild-type virus. The present studies indicate that attenuation is temporally regulated and suggest that agnoprotein enhances attenuation in isolated nuclei and that may also enhance it in vivo.

scholarly journals Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis

2009 ◽  
Vol 191 (15) ◽  
pp. 4916-4923 ◽  
Author(s):  
Chiaki Nakano ◽  
Hiroki Ozawa ◽  
Genki Akanuma ◽  
Nobutaka Funa ◽  
Sueharu Horinouchi
Keyword(s):  
Bacillus Subtilis ◽  
Polyketide Synthase ◽  
Bacterial Genome ◽  
Fatty Acyl ◽  
Gram Positive ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Vitro Analysis

ABSTRACT Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.

scholarly journals Examination of Campylobacter jejuni Putative Adhesins Leads to the Identification of a New Protein, Designated FlpA, Required for Chicken Colonization

2009 ◽  
Vol 77 (6) ◽  
pp. 2399-2407 ◽  
Author(s):  
Rebecca C. Flanagan ◽  
Jason M. Neal-McKinney ◽  
A. Singh Dhillon ◽  
William G. Miller ◽  
Michael E. Konkel
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Blot Hybridization ◽  
Broiler Chicks ◽  
Cell Adherence ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
Genes Encoding

ABSTRACT Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacterium's in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacterium's in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.

scholarly journals The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

1981 ◽  
Vol 193 (2) ◽  
pp. 541-552 ◽  
Author(s):  
L C Packman ◽  
W V Shaw
Keyword(s):  
Chloramphenicol Acetyltransferase ◽  
Alpha Subunit ◽  
Type I ◽  
Chloramphenicol Resistance ◽  
Type Iii ◽  
Naturally Occurring ◽  
Alpha Beta ◽  
Beta 2

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

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