scholarly journals Identification and Functional Analysis of Apoptotic Protease Activating Factor-1 (Apaf-1) from Spodoptera litura

Insects ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 64
Author(s):  
Haihao Ma ◽  
Xiumei Yan ◽  
Lin Yan ◽  
Jingyan Zhao ◽  
Jiping Song ◽  
...  
Keyword(s):  
Spodoptera Litura ◽  
Actinomycin D ◽  
Titration Calorimetry ◽  
Apoptosis Pathway ◽  
Downstream Effector ◽  
Lepidopteran Insects ◽  
Evolutionarily Conserved ◽  
Effector Caspases ◽  
And Function

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule, essential for activating initiator caspase and downstream effector caspases, which directly cause apoptosis. In fruit flies, nematodes, and mammals, Apaf-1 has been extensively studied. However, the structure and function of Apaf-1 in Lepidoptera remain unclear. This study identified a novel Apaf-1 from Spodoptera litura, named Sl-Apaf-1. Sl-Apaf-1 contains three domains: a CARD domain, as well as NOD and WD motifs, and is very similar to mammalian Apaf-1. Interference of Sl-apaf-1 expression in SL-1 cells blocked apoptosis induced by actinomycin D. Overexpression of Sl-apaf-1 significantly enhances apoptosis induced by actinomycin D in Sf9/SL-1/U2OS cells, suggesting that the function of Sl-Apaf-1 is evolutionarily conserved. Furthermore, Sl-Apaf-1 could interact with Sl-caspase-5 (a homologue of mammalian caspase-9) and yielded a binding affinity of 1.37 × 106 M–1 according isothermal titration calorimetry assay. Initiator caspase (procaspase-5) of S. litura could be activated by Sl-Apaf-1 (without WD motif) in vitro, and the activated Sl-caspase-5 could cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly caused apoptosis. This study demonstrates the key role of Sl-Apaf-1 in the apoptosis pathway, suggesting that the apoptosis pathway in Lepidopteran insects and mammals is conserved.

scholarly journals Immunohistochemical Characterization of Phosphorylated Ubiquitin in the Mouse Hippocampus

2020 ◽  
Author(s):  
Kosuke Kataoka ◽  
Andras Bilkei-Gorzo ◽  
Andreas Zimmer ◽  
Toru Asahi
Keyword(s):  
Cell Layer ◽  
Granule Cell Layer ◽  
Phosphatase And Tensin Homolog ◽  
Neuronal Markers ◽  
Tensin Homolog ◽  
Evolutionarily Conserved ◽  
Previous Hypothesis ◽  
Mouse Hippocampus ◽  
And Function

ABSTRACTMitochondrial autophagy (mitophagy) is an essential and evolutionarily conserved process that maintains mitochondrial integrity via the removal of damaged or superfluous mitochondria in eukaryotic cells. Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and Parkin promote mitophagy and function in a common signaling pathway. PINK1-mediated ubiquitin phosphorylation at Serine 65 (Ser65-pUb) is a key event in the efficient execution of PINK1/Parkin-dependent mitophagy. However, few studies have used immunohistochemistry to analyze Ser65-pUb in the mouse. Here, we examined the immunohistochemical characteristics of Ser65-pUb in the mouse hippocampus. Some hippocampal cells were Ser65-pUb positive, whereas the remaining cells expressed no or low levels of Ser65-pUb. PINK1 deficiency resulted in a decrease in the density of Ser65-pUb-positive cells, consistent with a previous hypothesis based on in vitro research. Interestingly, Ser65-pUb-positive cells were detected in hippocampi lacking PINK1 expression. The CA3 pyramidal cell layer and the dentate gyrus (DG) granule cell layer exhibited significant reductions in the density of Ser65-pUb-positive cells in PINK1-deficient mice. Moreover, Ser65-pUb immunoreactivity colocalized predominantly with neuronal markers. These findings suggest that Ser65-pUb may serve as a biomarker of in situ PINK1 signaling in the mouse hippocampus; however, the results should be interpreted with caution, as PINK1 deficiency downregulated Ser65-pUb only partially.

scholarly journals Role of Calcium in Protein Folding and Function of Tva, the Receptor of Subgroup A Avian Sarcoma and Leukosis Virus

2001 ◽  
Vol 75 (5) ◽  
pp. 2051-2058 ◽  
Author(s):  
Qing-Yin Wang ◽  
Klavs Dolmer ◽  
Wen Huang ◽  
Peter G. W. Gettins ◽  
Lijun Rong
Keyword(s):  
Protein Folding ◽  
Calcium Binding ◽  
Titration Calorimetry ◽  
Viral Receptor ◽  
Calcium Affinity ◽  
Binding Residues ◽  
And Function ◽  
Avian Sarcoma

ABSTRACT Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd ) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 μM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.

scholarly journals DNGR-1 is a specific and universal marker of mouse and human Batf3-dependent dendritic cells in lymphoid and nonlymphoid tissues

Blood ◽  
2012 ◽  
Vol 119 (25) ◽  
pp. 6052-6062 ◽  
Author(s):  
Lionel F. Poulin ◽  
Yasmin Reyal ◽  
Heli Uronen-Hansson ◽  
Barbara U. Schraml ◽  
David Sancho ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Transcription Factor ◽  
Human Blood ◽  
High Expression ◽  
Humanized Mice ◽  
Evolutionarily Conserved ◽  
And Function ◽  
Different Tissues

Abstract Mouse CD8α+ dendritic cells (DCs) in lymphoid organs and CD103+ CD11b− DCs in nonlymphoid tissues share phenotypic and functional similarities, as well as a unique shared developmental dependence on the transcription factor Batf3. Human DCs resembling mouse CD8α+ DCs in phenotype and function have been identified in human blood, spleen, and tonsil. However, it is not clear whether such cells are also present in human nonlymphoid organs, and their equivalence to mouse CD8α+ DC has recently been questioned. Furthermore, the identification of “CD8α+ DC-like” cells across different tissues and species remains problematic because of the lack of a unique marker that can be used to unambiguously define lineage members. Here we show that mouse CD8α+ DCs and CD103+ CD11b− DCs can be defined by shared high expression of DNGR-1 (CLEC9A). We further show that DNGR-1 uniquely marks a CD11b− human DC population present in both lymphoid and nonlymphoid tissues of humans and humanized mice. Finally, we demonstrate that knockdown of Batf3 selectively prevents the development of DNGR-1+ human DCs in vitro. Thus, high expression of DNGR-1 specifically and universally identifies a unique DC subset in mouse and humans. Evolutionarily conserved Batf3 dependence justifies classification of DNGR-1hi DCs as a distinct DC lineage.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

Type I Interferons promote maintenance and function of follicular T helper cells in vitro

2019 ◽  
Author(s):  
S Ehrlich ◽  
K Wild ◽  
M Smits ◽  
K Zoldan ◽  
M Hofmann ◽  
...  
Keyword(s):  
T Helper Cells ◽  
Type I Interferons ◽  
Type I ◽  
T Helper ◽  
Helper Cells ◽  
Follicular T Helper Cells ◽  
And Function

ANALYSIS OF THE BIPHASIC ACTION OF GROWTH HORMONE IN VITRO ON THE RAT DIAPHRAGM

1968 ◽  
Vol 57 (3_Suppl) ◽  
pp. S19-S35 ◽  
Author(s):  
Å. Hjalmarson
Keyword(s):  
Growth Hormone ◽  
Actinomycin D ◽  
Accumulation Rate ◽  
Inhibitory Effect ◽  
Bovine Growth Hormone ◽  
Rat Diaphragm ◽  
Dual Nature ◽  
Hypophysectomized Rats ◽  
D 20

ABSTRACT In vitro addition of bovine growth hormone (GH) to intact hemidiaphragms from hypophysectomized rats has previously been found to produce both an early stimulatory effect lasting for 2—3 hours and a subsequent late inhibitory effect during which the muscle is insensitive to further addition of GH (Hjalmarson 1968). These effects on the accumulation rate of α-aminoisobutyric acid (AIB) and D-xylose have been further studied. In presence of actinomycin D (20 μg/ml) or puromycin (100 μg/ml) the duration of the stimulatory effect of GH (25 μg/ml) was prolonged to last for at least 4—5 hours and the late inhibitory effect was prevented. Similar results were obtained when glucose-free incubation medium was used. Preincubation of the diaphragm at different glucose concentrations (0—5 mg/ml) for 3 hours did not change the GH sensitivity. Addition of insulin at start of incubation could not prevent GH from inducing its late inhibitory effect, while dexamethasone seemed to potentiate this effect of GH. Furthermore, adrenaline was found to decrease the uptake of AIB-14C and D-xylose-14C in the diaphragm, but not to change the sensitivity of the muscle to GH. Preincubation of the diaphragm for 3 hours with puromycin in a concentration of 200 μg/ml markedly decreased the subsequent basal uptake of both AIB-14C and D-xylose-14C, in the presence of puromycin, and abolished the stimulatory effect of GH on the accumulation of AIB-14C. However, the effect of GH on the accumulation of D-xylose-14C was unchanged. The present observations are discussed and evaluated in relation to various mechanisms of GH action proposed to explain the dual nature of the hormone.

CONTRIBUTION TO THE EXISTENCE OF REGULATORY MECHANISMS AT THE ADRENAL LEVEL

1972 ◽  
Vol 70 (4) ◽  
pp. 741-757
Author(s):  
Otto Linèt
Keyword(s):  
Orotic Acid ◽  
Adrenal Glands ◽  
Actinomycin D ◽  
Delay Period ◽  
Regulatory Mechanisms ◽  
Leucine Incorporation ◽  
Total Period ◽  
Free Media

ABSTRACT Rat adrenal glands atrophied by the administration of cortisol acetate in vivo were used as a model for the study of early metabolic processes occurring in vitro. Atrophied adrenals incubated in the presence of 14C-leucine incorporated subnormal quantities of this amino acid per mg of protein for the first 120 min. When the incubation lasted for a total period of 180 or 240 min a supranormal rise in the 14C-leucine incorporation was observed. Similar changes occurred with some delay with regard to corticosterone production as expressed per 100 mg of tissue. No differences in 14C-leucine incorporation were observed between the control and atrophied adrenals in vivo. Homogenates from atrophied glands incorporated 14C-leucine to a greater extent than the control homogenates. The in vitro incorporation of 14C-orotic acid into the RNA was also higher in atrophied adrenals. The in vitro use of actinomycin D, cycloheximide and amphenone indicated that corticosterone production depended on the incorporation of 14C-leucine. The addition of cortisol to the incubation media markedly decreased the enhancement of 14C-lysine incorporation into the protein of atrophied adrenals. These, as well as additional results suggest rebound phenomena: once atrophic adrenals are transferred to cortisol-free media, reparative processes begin after a delay period. Such phenomena seem to be mediated by regulatory mechanisms at the adrenal level.

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