scholarly journals Proadrenomedullin N-Terminal 20 Peptide Increases Kinesin's Velocity Both in Vitro and in Vivo

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1734-1742 ◽  
Author(s):  
Ignacio M. Larráyoz ◽  
Alfredo Martínez
Keyword(s):  
Cell Biology ◽  
Hippocampal Neurons ◽  
Motor Proteins ◽  
Time Lapse ◽  
Dependent Manner ◽  
Peptide Fragments ◽  
Motor Speed ◽  
Gene Coding

Intracellular cargo transport relies on microtubules and motor proteins such as kinesins and dyneins. Currently we have ample knowledge of the mechanisms by which motor proteins propel themselves along the microtubules, but little is known about intracellular factors that regulate motor speed. Here we show that proadrenomedullin N-terminal 20 peptide (PAMP) increases kinesin velocity and ATP consumption in a dose-dependent manner, using a variety of human kinesins. Structure-activity studies found that the terminal amide of PAMP is required for modulating kinesin activity and that the smallest peptide fragment retaining this role is PAMP(12–20). On the other hand, peptide fragments as small as PAMP(18–20) maintained the ability of delaying tubulin polymerization, another function previously described for PAMP, indicating that these two activities depend on different regions of the molecule. To demonstrate that these observations are also relevant in vivo, hippocampal neurons were isolated from mice lacking the gene coding for PAMP and from wild type littermates. Intravital stains followed by time-lapse microscopy analysis revealed that mitochondrial speed inside neurons lacking PAMP was significantly slower than in cells expressing the peptide. External addition of synthetic PAMP reversed this phenotype in PAMP-null neurons. Besides the obvious implications for better understanding cell biology, these results may be also relevant for the rapidly evolving discipline of nanotechnology because PAMP may be used as an accelerator of nanodevices based on microtubules and motor proteins.

scholarly journals A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions

2020 ◽  
Vol 295 (30) ◽  
pp. 10293-10306 ◽  
Author(s):  
Qiquan Wang ◽  
Xianling Bian ◽  
Lin Zeng ◽  
Fei Pan ◽  
Lingzhen Liu ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cell Biology ◽  
Biochemical Properties ◽  
Bacterial Toxin ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Membrane Pore ◽  
Intracellular Vesicles

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

scholarly journals Protective Effects of Scolopendra Water Extract on Trimethyltin-Induced Hippocampal Neurodegeneration and Seizures in Mice

2019 ◽  
Vol 9 (12) ◽  
pp. 369
Author(s):  
Yun-Soo Seo ◽  
Mary Jasmin Ang ◽  
Byeong Cheol Moon ◽  
Hyo Seon Kim ◽  
Goya Choi ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Primary Cultures ◽  
Water Extract ◽  
Model Systems ◽  
Inflammatory Factor ◽  
Dependent Manner ◽  
Protective Effects ◽  
Cell Degeneration

Trimethyltin (TMT) is an organotin compound with potent neurotoxic action characterized by neuronal degeneration in the hippocampus. This study evaluated the protective effects of a Scolopendra water extract (SWE) against TMT intoxication in hippocampal neurons, using both in vitro and in vivo model systems. Specifically, we examined the actions of SWE on TMT- (5 mM) induced cytotoxicity in primary cultures of mouse hippocampal neurons (7 days in vitro) and the effects of SWE on hippocampal degeneration in adult TMT- (2.6 mg/kg, intraperitoneal) treated C57BL/6 mice. We found that SWE pretreatment (0–100 μg/mL) significantly reduced TMT-induced cytotoxicity in cultured hippocampal neurons in a dose-dependent manner, as determined by lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. Additionally, this study showed that perioral administration of SWE (5 mg/kg), from −6 to 0 days before TMT injection, significantly attenuated hippocampal cell degeneration and seizures in adult mice. Furthermore, quantitative analysis of Iba-1 (Allograft inflammatory factor 1)- and GFAP (Glial fibrillary acidic protein)-immunostained cells revealed a significant reduction in the levels of Iba-1- and GFAP-positive cell bodies in the dentate gyrus (DG) of mice treated with SWE prior to TMT injection. These data indicated that SWE pretreatment significantly protected the hippocampus against the massive activation of microglia and astrocytes elicited by TMT. In addition, our data showed that the SWE-induced reduction of immune cell activation was linked to a significant reduction in cell death and a significant improvement in TMT-induced seizure behavior. Thus, we conclude that SWE ameliorated the detrimental effects of TMT toxicity on hippocampal neurons, both in vivo and in vitro. Altogether, our findings hint at a promising pharmacotherapeutic use of SWE in hippocampal degeneration and dysfunction.

Imaging Molecular Targeting In Living Neural Cultures

1999 ◽  
Vol 5 (S2) ◽  
pp. 1228-1229
Author(s):  
Christopher S. Wallace ◽  
Michael A. Silverman ◽  
Michelle A. Burack ◽  
Janis E. Lochner ◽  
Richard G. Allen ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Hippocampal Neurons ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Complex Structure ◽  
Time Lapse ◽  
The Central Nervous System

Recent technical advances in the ability to attach an endogenously fluorescent protein sequence—i.e., green fluorescent protein or GFP and its derivatives--to any protein of experimental interest promises to mark a new era of progress in the study of protein targeting. Bringing these new tools to bear on neurons of the central nervous system has been challenging, however, because they have a very complex structure and are relatively difficult to transfect because they are post-mitotic.We use two cell culture approaches to characterize protein trafficking within neurons of the central nervous system in vitro. The first is a dissociated culture of hippocampal neurons from embryonic (El8) rats which is especially suited to analysis by conventional light microscopy because these neurons are grown on glass coverslips at low density. Neurons cultured in this way develop a morphology comparable to that seen in vivo and permit the establishment of axons and dendrites to be analyzed by time-lapse microscopy.

scholarly journals Imaging cell biology in live animals: Ready for prime time

2013 ◽  
Vol 201 (7) ◽  
pp. 969-979 ◽  
Author(s):  
Roberto Weigert ◽  
Natalie Porat-Shliom ◽  
Panomwat Amornphimoltham
Keyword(s):  
Cell Biology ◽  
Cancer Biology ◽  
In Vitro Model ◽  
Time Lapse ◽  
Living Cells ◽  
Model Systems ◽  
Prime Time ◽  
Vitro Model

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.

scholarly journals Red Ginseng Extract Attenuates Kainate-Induced Excitotoxicity by Antioxidative Effects

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Jin-Yi Han ◽  
Sun-Young Ahn ◽  
Eun-Hye Oh ◽  
Sang-Yoon Nam ◽  
Jin Tae Hong ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Mossy Fiber ◽  
Neuroprotective Effect ◽  
Order Rate Constant ◽  
Neuroprotective Activity ◽  
Dependent Manner ◽  
Red Ginseng ◽  
Ginseng Extract

This study investigated the neuroprotective activity of red ginseng extract (RGE,Panax ginseng, C. A. Meyer) against kainic acid- (KA-) induced excitotoxicityin vitroandin vivo. In hippocampal cells, RGE inhibited KA-induced excitotoxicity in a dose-dependent manner as measured by the MTT assay. To study the possible mechanisms of the RGE-mediated neuroprotective effect against KA-induced cytotoxicity, we examined the levels of intracellular reactive oxygen species (ROS) and [Ca2+]iin cultured hippocampal neurons and found that RGE treatment dose-dependently inhibited intracellular ROS and [Ca2+]ielevation. Oral administration of RGE (30 and 200 mg/kg) in mice decreased the malondialdehyde (MDA) level induced by KA injection (30 mg/kg, i.p.). In addition, similar results were obtained after pretreatment with the radical scavengers Trolox andN,N′-dimethylthiourea (DMTU). Finally, after confirming the protective effect of RGE on hippocampal brain-derived neurotropic factor (BDNF) protein levels, we found that RGE is active compounds mixture in KA-induced hippocampal mossy-fiber function improvement. Furthermore, RGE eliminated 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and the IC50was approximately 10 mg/ml. The reductive activity of RGE, as measured by reaction with hydroxyl radical (•OH), was similar to trolox. The second-order rate constant of RGE for•OH was 3.5–4.5×109 M−1·S−1. Therefore, these results indicate that RGE possesses radical reduction activity and alleviates KA-induced excitotoxicity by quenching ROS in hippocampal neurons.

scholarly journals The Effects of Yuan-Zhi Decoction and Its Active Ingredients in Both In Vivo and In Vitro Models of Chronic Cerebral Hypoperfusion by Regulating the Levels of Aβ and Autophagy

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Yan Liu ◽  
Xiaobo Huang ◽  
Wenqiang Chen ◽  
Yujing Chen ◽  
Ningqun Wang ◽  
...  
Keyword(s):  
Cognitive Function ◽  
Hippocampal Neurons ◽  
The Elderly ◽  
Chronic Cerebral Hypoperfusion ◽  
Cerebral Hypoperfusion ◽  
Dependent Manner ◽  
Active Ingredients ◽  
Related Proteins

Chronic cerebral hypoperfusion (CCH) is closely related to the occurrence of Alzheimer’s disease (AD) in the elderly. CCH can induce overactivation of autophagy, which increases the deposition of amyloid-β (Aβ) plaques in the brain, eventually impairing the cognitive function. Yuan-Zhi decoction (YZD) is a traditional Chinese medicine (TCM) formulation that is used to treat cognitive dysfunction in the elderly, but the specific mechanism is still unclear. In this study, we simulated CCH in a rat model through bilateral common carotid artery occlusion (BCCAO) and treated the animals with YZD. Standard neurological tests indicated that YZD significantly restored the impaired cognitive function after BCCAO in a dose-dependent manner. Furthermore, YZD also decreased the levels of Aβ aggregates and the autophagy-related proteins ATG5 and ATG12 in their hippocampus. An in vitro model of CCH was also established by exposing primary rat hippocampal neurons to hypoxia and hypoglycemia (H-H). YZD and its active ingredients increased the survival of these neurons and decreased the levels of Aβ1-40 and Aβ1-42, autophagy-related proteins Beclin-1 and LC3-II, and the APP secretases BACE1 and PS-1. Finally, both Aβ aggregates showed a positive statistical correlation with the expression levels of the above proteins. Taken together, YZD targets Aβ, autophagy, and APP-related secretases to protect the neurons from hypoxic-ischemic injury and restore cognitive function.

scholarly journals Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

2008 ◽  
Vol 19 (11) ◽  
pp. 4804-4813 ◽  
Author(s):  
Haimin Li ◽  
Gang Chen ◽  
Bing Zhou ◽  
Shumin Duan
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Time Lapse ◽  
Dependent Manner ◽  
Filamentous Actin ◽  
Kinase Substrate ◽  
Extensive Growth ◽  
Time Lapse Imaging

Dendrites undergo extensive growth and branching at early stages, but relatively little is known about the molecular mechanisms underlying these processes. Here, we show that increasing the level of myristoylated, alanine-rich C kinase substrate (MARCKS), a prominent substrate of protein kinase C and a phosphatidylinositol-4,5-diphosphate [PI(4,5)P2] sequestration protein highly expressed in the brain, enhanced branching and growth of dendrites both in vitro and in vivo. Conversely, knockdown of endogenous MARCKS by RNA interference reduced dendritic arborization. Results from expression of different mutants indicated that membrane binding is essential for MARCKS-induced dendritic morphogenesis. Furthermore, MARCKS increased the number and length of filamentous actin-based filopodia along neurites, as well as the motility of filopodia, in a PI(4,5)P2-dependent manner. Time-lapse imaging showed that MARCKS increased frequency of filopodia initiation but did not affect filopodia longevity, suggesting that MARCKS may increase dendritic branching through its action on filopodia initiation. These findings demonstrate a critical role for MARCKS–PI(4,5)P2 signaling in regulating dendrite development.

scholarly journals Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion

2016 ◽  
Vol 213 (9) ◽  
pp. 1881-1900 ◽  
Author(s):  
Martin Chopra ◽  
Marlene Biehl ◽  
Tim Steinfatt ◽  
Andreas Brandl ◽  
Juliane Kums ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Biology ◽  
Acute Gvhd ◽  
Ex Vivo ◽  
Tumor Necrosis Factor Receptor ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Study Protocols

Donor CD4+Foxp3+ regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell–dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

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