scholarly journals Immunolocalization of lipoxygenase in the microspore of Gagea lutea (L.) Ker.-Gaw.

2012 ◽  
Vol 59 (1) ◽  
pp. 83-90
Author(s):  
Ewa Szczuka ◽  
Bożena Pawlikowska-Pawlęga ◽  
Ewa Skórzyńska-Polit ◽  
Jolanta Sobieska ◽  
Jarosław Pawelec ◽  
...  
Keyword(s):  
Immunogold Labelling ◽  
Gagea Lutea ◽  
Labelling Method

Localization of lipoxygenase (LOX) in the microspore of <i>Gagea lutea</i> (L.) Ker.-Gaw. was investigated with the immunogold labelling method. The enzyme was found in the cytoplasm, nucleus and sporoderm. The most intensive reaction was observed in the cytoplasm, where the immunogold particles were sometimes grouped into clusters of several or more and showed the highest density. The smallest amount of particles occured in the sporoderm. The role of lipoxygenase in the microspore is discussed.

scholarly journals Crash landing of Vibrio cholerae by MSHA pili-assisted braking and anchoring in a viscous environment

2020 ◽  
Author(s):  
Wenchao Zhang ◽  
Mei Luo ◽  
Chunying Feng ◽  
Rachel R. Bennett ◽  
Andrew S. Utada ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Cell Tracking ◽  
Surface Attachment ◽  
Near Surface ◽  
Cysteine Substitution ◽  
Resistive Force Theory ◽  
Viscous Solutions ◽  
Labelling Method ◽  
Detailed Picture

AbstractMannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of Vibrio cholerae. However, the cell landing mechanism remains largely unknown. Here, combining the cysteine-substitution-based labelling method with single-cell tracking techniques, we quantitatively characterized the landing of V. cholerae by directly observing both pili and flagellum of cells in viscous solutions. MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which include three phases: running, lingering, and attaching. Resistive-force-theory based models are proposed to describe near-surface motion. Importantly, the role of MSHA pili during cell landing is more apparent in viscous solutions. Our work provides a detailed picture of the landing dynamics of V. cholerae under viscous conditions, which can provide insights into ways to better control V. cholerae infections.

scholarly journals An immunogold labelling method for the enumeration of canine T‐lymphocytes

1988 ◽  
Vol 10 (4) ◽  
pp. 273-276 ◽  
Author(s):  
G. O. Evans ◽  
R. M. Flynn ◽  
J. D. Lupton
Keyword(s):  
T Lymphocytes ◽  
Immunogold Labelling ◽  
Labelling Method

scholarly journals Regulation of photosynthetic electron flow on dark to light transition by ferredoxin:NADP(H) oxidoreductase interactions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Manuela Kramer ◽  
Melvin Rodriguez-Heredia ◽  
Francesco Saccon ◽  
Laura Mosebach ◽  
Manuel Twachtmann ◽  
...  
Keyword(s):  
Electron Transport ◽  
Electron Flow ◽  
Carbon Assimilation ◽  
Photosynthetic Electron Transport ◽  
Immunogold Labelling ◽  
Free Radical Damage ◽  
Transport Pathways ◽  
Photosynthetic Electron Flow ◽  
Radical Damage

During photosynthesis, electron transport is necessary for carbon assimilation and must be regulated to minimize free radical damage. There is a longstanding controversy over the role of a critical enzyme in this process (ferredoxin:NADP(H) oxidoreductase, or FNR), and in particular its location within chloroplasts. Here we use immunogold labelling to prove that FNR previously assigned as soluble is in fact membrane associated. We combined this technique with a genetic approach in the model plant Arabidopsis to show that the distribution of this enzyme between different membrane regions depends on its interaction with specific tether proteins. We further demonstrate a correlation between the interaction of FNR with different proteins and the activity of alternative photosynthetic electron transport pathways. This supports a role for FNR location in regulating photosynthetic electron flow during the transition from dark to light.

A structural approach to pathological crystallizations. Gout: the possible role of albumin in sodium urate crystallization

1988 ◽  
Vol 235 (1279) ◽  
pp. 145-159 ◽  
Keyword(s):  
Crystal Morphology ◽  
Protein Denaturation ◽  
Immunogold Labelling ◽  
Sodium Cation ◽  
Carboxylate Groups ◽  
Sodium Urate ◽  
Msu Crystals ◽  
Nucleating Effect

The interactions between sodium urate monohydrate (MSU) crystals and human serum albumin (HSA) were investigated in vitro in relation to the disease of gout. It was found that HSA accelerates (by up to ten times or even more) the nucleation of MSU crystals at a pH of more than 7.5, but only to a much lesser extent (1.2 times) at pH 7.0. Protein denaturation, as well as blocking exposed carboxylate groups on the protein, substantially reduced the nucleating effect. By use of immunofluorescence, immunogold labelling and crystal morphology studies, albumin was shown to interact preferentially with the {11̄0} faces of MSU crystals. Taking these results into consideration, a mechanism is proposed whereby albumin stabilizes MSU crystal nuclei by interaction of structured carboxylate-containing protein domains with planes of the incipient crystal exposing sodium cation layers.

The ordered macromolecular surface of polyester inclusion bodies inPseudomonas oleovotans

1995 ◽  
Vol 41 (13) ◽  
pp. 84-93 ◽  
Author(s):  
Elizabeth S. Stuart ◽  
R. Clinton Fuller ◽  
Robert W. Lenz
Keyword(s):  
Inclusion Bodies ◽  
Amorphous State ◽  
Immunogold Labelling ◽  
Boundary Region ◽  
Pseudomonas Oleovorans ◽  
Sds Page ◽  
Degradative Enzymes ◽  
Layer Chromatography ◽  
Intracellular Inclusion

Intracellular inclusion bodies of poly(β-hydroxyalkanoates) (PHAs) have been studied in various microorganisms since Lemoigne's discovery of PHAs in 1925. Recently, the research in several laboratories, including our own, has addressed the role of proteins, lipids, and water associated with these accumulations. Our research has examined the boundary of polymer inclusion bodies. Electron microscopy demonstrated that the polymer is encompassed by two paracrystalline arrays. SDS-PAGE, Western blot, or immunogold labelling demonstrated that both contain a 43-kDa protein as a major component. Immunogold labelling also demonstrated that 55- and 59-kDa proteins are located, exclusively, on the array associated with the accumulating polymer. Results from microelemental analysis and preliminary thin-layer chromatography of released lipids were consistent with the suggestion that phospholipids also had a role in this organized assembly. A model has been suggested, aimed at focusing attention on this organized boundary region. It is consistent with maintenance of the amorphous state of the polymer both intracellularly and after isolation, provides sites for biosynthetic and degradative enzymes, and accounts for the polyester, protein, and lipid components known to be present. Interestingly, the anti-43-kDa antibody also recognized a 43-kDa species released from the outer surface of this microbe. The research presented here and the model developed from it, suggest that microbial synthesis, containment, and degradation of polyester are carried out in association with a highly organized and complex intracellular assembly that may provide, within the bacterial cytosol, a unique microenvironment for biochemical activities.Key words: polyester, inclusion granule, Pseudomonas oleovorans.

Ultrastructural Analysis of Megakaryocytes in GPV Knockout Mice

2000 ◽  
Vol 84 (08) ◽  
pp. 312-318 ◽  
Author(s):  
Christel Poujol ◽  
Vanitha Ramakrishnan ◽  
Francis DeGuzman ◽  
Alan Nurden ◽  
David Phillips ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Knockout Mice ◽  
Platelet Adhesion ◽  
Membrane System ◽  
Immunogold Labelling ◽  
Bleeding Diathesis ◽  
Giant Platelets ◽  
Negative Modulator ◽  
Bernard Soulier Syndrome

SummaryLesions in the genes for GPIbα, GPIbβ or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.

scholarly journals Pathological Features of Experimental Bovine Leukaemia Viral (BLV) Infection in Rats and Rabbits

2012 ◽  
Vol 56 (2) ◽  
pp. 115-120
Author(s):  
Petar Dimitrov ◽  
Konstantin Simeonov ◽  
Katerina Todorova ◽  
Zina Ivanova ◽  
Reneta Toshkova ◽  
...  
Keyword(s):  
Immunogold Labelling ◽  
Laboratory Animals ◽  
Aetiological Factor ◽  
Pcr Analysis ◽  
Bovine Leukaemia Virus ◽  
Leukaemia Virus ◽  
Tax Protein ◽  
Transmission Electron

Abstract Rabbits and rats were inoculated with material derived from FLK cells producing permanently bovine leukaemia virus (BLV). The viral presence in the inoculum was proved by transmission electron microscopy, immunofluorescence, immunogold labelling demonstrating viral Tax protein, and PCR analysis. About 30 % of the infected animals sustained BLV seropositivity during the experiment, and demonstrated symptoms of lympholeukaemia - clinical manifestation of an immunosuppressive condition, increased number of lymphocytes and lymphoblasts, and preneoplastic lymphoid cell accumulations in the liver, lungs, kidneys, and lymph nodes. BLV DNA, detected by PCR in diseased animals, indicates the role of BLV as an aetiological factor of lympholeukaemia, developed in these animals after BLV infection. The alterations in rats were more pronounced than those in rabbits. The results prove that these two species of laboratory animals, especially rats, are suitable models for the in vivo studies of leukaemogenesis caused by BLV/HTLV infections.

Trypanosoma congolense: appearance and distribution of variable antigen types during metacyclic differentiation in vitro

Parasitology ◽  
1990 ◽  
Vol 100 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C. J. Prain ◽  
C. A. Ross
Keyword(s):  
Culture System ◽  
Antibody Test ◽  
Immunogold Labelling ◽  
Trypanosoma Congolense ◽  
Attachment Sites ◽  
Variable Antigen ◽  
Labelling Method ◽  
Immunofluorescence Antibody Test ◽  
Immunofluorescence Antibody

SummaryDifferentiation of epimastigotes and production of infective metacyclic forms of Trypanosoma congolense were examined in a culture system which enabled manipulation of the population density of insect forms. Scanning electron microscopy of cultures revealed the attachment sites of epimastigotes in detail, showing them to be attached as ‘clusters’ or ‘bundles’ and having associated fibrillar structures. Dividing epimastigotes were observed either within individual bundles or in association with two bundles. Metacyclic forms were detected by an immunofluorescence antibody test (IFAT) using metacyclic variable-antigen type (M-VAT) specific monoclonal antibodies, by day 7 after seeding cultures. Trypanosomes expressing M-VATs appeared singly in bundles, observed by both IFAT and an immunogold labelling method. Statistical analysis using Poisson calculations suggested that, in general, the distribution of metacyclics expressing individual M-VATs was random throughout cultures.

scholarly journals An activity-based labelling method for the detection of ammonia and methane-oxidizing bacteria

2021 ◽  
Author(s):  
Dimitra Sakoula ◽  
Garrett J. Smith ◽  
Jeroen Frank ◽  
Rob J. Mesman ◽  
Linnea F.M. Kop ◽  
...  
Keyword(s):  
Cycloaddition Reaction ◽  
Immunogold Labelling ◽  
Protein Profiling ◽  
Microbial Biodiversity ◽  
Carbon Cycles ◽  
Methane Oxidizing Bacteria ◽  
Phylogenetic Identification ◽  
Biotin Labelling ◽  
Labelling Method

AbstractThe advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia- and methane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. However, the in situ detection and phylogenetic identification of novel ammonia- and methane-oxidizing bacteria remains a challenge. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and methane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labelling of cells harboring active ammonia and methane monooxygenases. The biotinylation of these enzymes in combination with immunogold labelling reveals the subcellular localization of the tagged proteins, while the fluorescent labelling of cells harboring active ammonia or methane monooxygenases provides a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labelling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and methanotrophs from complex environmental samples, facilitating the retrieval of their high quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and methane-oxidizing bacteria present in complex microbial communities.

scholarly journals Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores.

2021 ◽  
Author(s):  
Marjorie Pizarro-Guajardo ◽  
Cesar Ortega-Lizarraga ◽  
Ana Inostroza-Mora ◽  
Francisca Cid-Rojas ◽  
Daniel Paredes-Sabja
Keyword(s):  
Immunogold Labelling ◽  
Sigma Factors ◽  
Surface Proteins ◽  
Dense Layer ◽  
Spore Surface ◽  
Wild Type ◽  
Susceptible Host ◽  
Clostridioides Difficile ◽  
In The Wild

Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.

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