scholarly journals Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

2007 ◽  
Vol 402 (2) ◽  
pp. 229-239 ◽  
Author(s):  
Daniela B. Munafó ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Sophie Rutschmann ◽  
Bruce Beutler ◽  
...  
Keyword(s):  
Immunofluorescence Microscopy ◽  
Small Gtpase ◽  
Effector Protein ◽  
Cell Fractionation ◽  
Density Fraction ◽  
Fractionation Analysis ◽  
Minor Population ◽  
A Minor ◽  
Micro Organisms ◽  
Deficient Mice

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1–Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway

1995 ◽  
Vol 108 (11) ◽  
pp. 3509-3521 ◽  
Author(s):  
B. Singer-Kruger ◽  
H. Stenmark ◽  
M. Zerial
Keyword(s):  
Small Gtpase ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Cell Fractionation ◽  
Animal Cells ◽  
Early Endosomes ◽  
Fractionation Analysis ◽  
Alpha Factor ◽  
Vacuolar Protein

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM

2020 ◽  
Author(s):  
MAK Williams ◽  
V Cornuault ◽  
AH Irani ◽  
VV Symonds ◽  
J Malmström ◽  
...  
Keyword(s):  
Average Length ◽  
American Chemical Society ◽  
Md Simulations ◽  
Chemical Society ◽  
Side Chains ◽  
Rhamnogalacturonan I ◽  
Afm Imaging ◽  
Minor Population ◽  
The Stability ◽  
A Minor

© 2020 American Chemical Society. Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

scholarly journals IL-6 Is Not Absolutely Essential for the Development of a TH17 Immune Response after an Aerosol Infection with Mycobacterium tuberculosis H37rv

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 9
Author(s):  
Kristina Ritter ◽  
Jan Christian Sodenkamp ◽  
Alexandra Hölscher ◽  
Jochen Behrends ◽  
Christoph Hölscher
Keyword(s):  
Immune Response ◽  
Inflammatory Diseases ◽  
Mycobacterial Infection ◽  
Minor Effect ◽  
Mycobacterium Tuberculosis H37rv ◽  
Th 17 ◽  
Anti Inflammatory ◽  
A Minor ◽  
Aerosol Infection ◽  
Deficient Mice

Anti-inflammatory treatment of chronic inflammatory diseases often increases susceptibility to infectious diseases such as tuberculosis (TB). Since numerous chronic inflammatory and autoimmune diseases are mediated by interleukin (IL)-6-induced T helper (TH) 17 cells, a TH17-directed anti-inflammatory therapy may be preferable to an IL-12-dependent TH1 inhibition in order to avoid reactivation of latent infections. To assess, however, the risk of inhibition of IL-6-dependent TH17-mediated inflammation, we examined the TH17 immune response and the course of experimental TB in IL-6- and T-cell-specific gp130-deficient mice. Our study revealed that the absence of IL-6 or gp130 on T cells has only a minor effect on the development of antigen-specific TH1 and TH17 cells. Importantly, these gene-deficient mice were as capable as wild type mice to control mycobacterial infection. Together, in contrast to its key function for TH17 development in other inflammatory diseases, IL-6 plays an inferior role for the generation of TH17 immune responses during experimental TB.

Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes

1990 ◽  
Vol 97 (1) ◽  
pp. 11-21
Author(s):  
M. Vitadello ◽  
M. Matteoli ◽  
L. Gorza
Keyword(s):  
Subcellular Distribution ◽  
Ultrastructural Level ◽  
Rabbit Heart ◽  
Cardiac Cells ◽  
Structural Components ◽  
Minor Population ◽  
Neuronal Proteins ◽  
Tissue Homogenates ◽  
A Minor

We have recently shown that specialized myocytes of the rabbit heart express a cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this result was obtained using a single anti-NF-M monoclonal antibody, we tested on conduction myocytes a panel of five anti-NF antibodies, specific for each of the three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two antibodies, one specific for the L subunit and one for phosphorylated M subunit of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots on conduction tissue homogenates the two antibodies recognized two polypeptides with electrophoretic mobility and solubility properties identical to those of NF-L and NF-M in the sciatic nerve. The subcellular distribution of NF immunoreactivity in specialized myocytes was very similar to desmin localization; namely, it was distributed on large filamentous bundles and on fine filaments localized transversely at the level of the Z line. At the ultrastructural level, immunoreactive filaments were localized in the intermyofibrillar space and connected myofibrils with mitochondria. Co-expression of NF proteins and desmin was also observed in vitro in a minor population of cardiac myocytes cultured from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with desmin, especially where filaments were well organized, but in some cells anti-NF and anti-desmin antibodies labelled different filamentous structures. These results indicate that NF proteins are structural components of the cytoskeleton of specialized myocytes and show a subcellular distribution very similar to desmin. Such a composition of intermediate filaments indicates that in these cardiac cells muscle differentiation is compatible with the expression of neuronal proteins.

Abstract 311: Measurements of cAMP Dynamics in the SERCA2 Microdomain in Adult Mouse Cardiomyocytes using a Targeted FRET Biosensor

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Julia U Sprenger ◽  
Viacheslav O Nikolaev
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Heart Weight ◽  
Cell Fractionation ◽  
Adrenergic Stimulation ◽  
Cardiac Phenotype ◽  
Fractionation Analysis

PURPOSE: cAMP is a central regulator of cardiac function and disease. This global second messenger acts in a compartmentalized fashion, and changes in cAMP dynamics are linked to cardiac diseases. In this project, we visualized cAMP signals directly in such microdomains to gain insights into the molecular mechanisms involved in cAMP compartmentation and its alterations in hypertrophy. Methods: We generated transgenic mice expressing a new Förster resonance energy transfer (FRET)-based cAMP sensor Epac1-camps-PLN to measure cAMP dynamics in the microdomain around the sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2). This sensor is targeted to SERCA2 via phospholamban (PLN). Results: Colocalization and cell fractionation analysis confirmed proper localization of the sensor in transgenic mouse hearts. qPCR analysis revealed a two-fold overexpression of PLN. However, no adverse cardiac phenotype could be detected by histological analysis and heart weight to body weight ratios. Local cAMP dynamics were measured using freshly isolated adult ventricular myocytes and compared to cAMP signals in the bulk cytosol using cardiomyocytes from Epac1-camps mice. We detected the predominant role of phosphodiesterases (PDEs) 4 and 3 in the SERCA2 compartment under basal conditions. These PDEs were responsible for shaping the microdomain and its segregation from the cytosolic compartment. Interestingly, beta1-adrenergic stimulation led to a stronger increase of local cAMP in the SERCA2 compartment compared to the bulk cytosol. 8 weeks after transverse aortic constriction (TAC), PDE4 activity was downregulated in the SERCA2 microdomain compared to sham cardiomyocytes. Conclusion: We successfully generated transgenic mice expressing the targeted Epac1-camps-PLN biosensor to visualize cAMP dynamics in the SERCA2 compartment. We could show distinct cAMP dynamics around the SERCA2 compartment compared to the bulk cytosol and uncovered its alterations in hypertrophied cardiomyocytes

scholarly journals Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis

2020 ◽  
Vol 21 (2) ◽  
pp. 644 ◽  
Author(s):  
Eva B. Znalesniak ◽  
Franz Salm ◽  
Werner Hoffmann
Keyword(s):  
Cysteine Residue ◽  
Molecular Forms ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Rt Pcr ◽  
Molecular Alterations ◽  
Protein Levels ◽  
Increased Sensitivity ◽  
A Minor ◽  
Deficient Mice

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

scholarly journals The basic study on kappa-lambda imaging by delta-curve for the detection of a monoclonal B-cell population in the peripheral blood

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1461-1466 ◽  
Author(s):  
M Nakano ◽  
S Kuge ◽  
S Kuwabara ◽  
M Yaguchi ◽  
Y Kawanishi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Basic Study ◽  
Minor Population ◽  
D Values ◽  
A Minor ◽  
D Value

Abstract Recently, kappa-lambda analysis with the “D” value was developed by Ault to detect a minor population of malignant B cells in peripheral blood. This analysis is based on the Kolmogorov-Smirnov test, and the D value is calculated by a flowcytometer and a computer. We have recently devised a more sensitive parameter for the kappa-lambda analysis than the D value called the delta-curve (delta c); the delta c applies the same principle as that of the D value. Mixing experiments with kappa- type and lambda-type chronic lymphocytic leukemia cells revealed that the delta c could not only detect a minor population of malignant kappa- B cells, but also that of malignant lambda-B cells using more sensitivity than the D value. A total of 49 blood samples obtained from 27 patients with various B-cell malignancies were investigated. D values were abnormal in 37% of all samples, while abnormal patterns of the delta c were recognized in 71%. On the other hand, 59% of samples obtained from the patients with B-cell lymphoma in aleukemic phase showed abnormal delta c, whereas D values exceeded the upper limit of the normal value in only 15% of the samples. It was suggested that the delta c could detect 3% to 7% of malignant B cells that were mixed with a population of normal lymphocytes.

scholarly journals A minor population of macrophage-tropic HIV-1 variants is identified in recrudescing viremia following analytic treatment interruption

2020 ◽  
Vol 117 (18) ◽  
pp. 9981-9990 ◽  
Author(s):  
Viviane M. Andrade ◽  
Carla Mavian ◽  
Dunja Babic ◽  
Thaissa Cordeiro ◽  
Mark Sharkey ◽  
...  
Keyword(s):  
Treatment Interruption ◽  
Viral Reservoir ◽  
Analytic Treatment ◽  
Recombinant Viruses ◽  
Minor Population ◽  
Clock Analysis ◽  
A Minor ◽  
Molecular Clock Analysis ◽  
Insight Into ◽  
Hiv 1

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses constructed with individual envelope genes that were obtained from plasma of six individuals undergoing analytic treatment interruption (ATI). M-tropic viruses could also be enriched from post-ATI plasma using macrophage-specific (CD14) but not CD4+ T cell-specific (CD3) antibodies, suggesting that M-tropic viruses had a macrophage origin. Molecular clock analysis indicated that the establishment of M-tropic HIV-1 variants predated ATI. Collectively, these data suggest that macrophages are a viral reservoir in HIV-1–infected individuals on effective ART and that M-tropic variants can appear in rebounding viremia when treatment is interrupted. These findings have implications for the design of curative strategies for HIV-1.

scholarly journals The Role ofγδT Cells in Systemic Lupus Erythematosus

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Meng Wu ◽  
Jinhua Yang ◽  
Xiaofeng Li ◽  
Junwei Chen
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
Autoimmune Disease ◽  
Lupus Erythematosus ◽  
Systemic Lupus ◽  
Multiple Organs ◽  
Minor Population ◽  
Neuronal Tissues ◽  
A Minor

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the overproduction of autoantibodies against an array of nuclear and cytoplasmic antigens and affects multiple organs, such as the skin, joints, kidneys, and neuronal tissues. T cells have been recognized as important players in the development of SLE due to their functions in cytokine secretion, antigen presentation, and supporting B cells for antibody production.γδT cells are a minor population of T cells that play important roles in infection and tumor-associated disease. In recent years, the role ofγδT cells in autoimmune diseases has been investigated. In this review, we discussed the role ofγδT cells in the pathogenesis of SLE.

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