1.Purification of clathrin assembly protein from rat liver.
Experimental & Molecular Medicine 2000;32(4):222-226
Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.
Animal
;
Clathrin/*metabolism
;
Clathrin-Coated Vesicles/*chemistry
;
Liver/*chemistry
;
Nerve Tissue Proteins/*isolation & purification
;
Phosphoproteins/*isolation & purification
;
Rats
2.Mechanism of cellular uptake and transport mediated by integrin receptor targeting trimethyl chitosan nanoparticles.
Juan XU ; Chong LIU ; Yi-ning XU ; Wei SHAN ; Min LIU ; Yuan HUANG
Acta Pharmaceutica Sinica 2015;50(7):893-898
This study investigated a nano drug delivery system built by one sort of modified trimethyl chitosan (TMC). The TMC was modified by cRGDyk, ligand of integrin receptor avβ3. Single factor screening was used to optimize the prescription in which the particle sizes of TMC nanoparticle (TMC NPs) and cRGDyk modified TMC nanoparticle (C-TMC NPs) were (240.3 ± 4.2) nm and (259.5 ± 3.3) nm. Electric potential of those two nanoparticles were (33.5 ± 0.8) mV and (25.7 ± 1.6) mV. Encapsulation efficiencies were (76.0 ± 2.2) % and (74.4 ± 2.0) %. Drug loading efficacies were (50.1 ± 2.1) % and (26.1 ± 1.0) %. Then the cellular uptake, uptake mechanism and transport efficacy of TMC NPs and C-TMC NPs were investigated using Caco-2 cell line. The uptake rate and accumulating drug transit dose of C-TMC NPs were 1.98 and 2.84 times higher than TMC NPs, separately. Mechanism investigations revealed that caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis were involved in the intercellular uptake of both TMC NPs and C-TMC NPs. What is more, free cRGDyk could remarkably inhibit the uptake of C-TMC NPs.
Biological Transport
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Caco-2 Cells
;
Caveolae
;
Chitosan
;
chemistry
;
Clathrin
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Endocytosis
;
Humans
;
Integrin alphaVbeta3
;
chemistry
;
Nanoparticles
;
Particle Size
;
Pinocytosis
3.Recent advances in the study of synaptic endocytosis key protein: Dynamin.
Journal of Central South University(Medical Sciences) 2014;39(10):1088-1092
As the basic physiological function of synapses, vesicle cycling involves in many aspects of process. Among them, vesicle recycling is the basis of synaptic vesicle cycling. Studies show that clathrin mediated endocytosis is a major pathway of vesicle recycling, in which Dynamin plays an important role. Dynamin is a GTPases with molecular weight of 100 kD, which acts as "scissors" in the endocytosis, separating the clathrin coated pits from membrane. It has been found that Dynamin is associated with epilepsy, Alzheimer's disease, centronuclear myopathy, and several other neurological diseases. In this paper, we discussed the structure, function and regulation of Dynamin, and reviewed recent advance in the studies on Dynamin related diseases.
Clathrin
;
physiology
;
Coated Pits, Cell-Membrane
;
physiology
;
Dynamins
;
physiology
;
Endocytosis
;
Humans
;
Synapses
;
physiology
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Synaptic Transmission
;
Synaptic Vesicles
;
physiology
4.Changes in expression of synaptic proteins in brains of patients with Alzheimer's disease.
Ying CAO ; Rivka RAVID ; Zhi-zhong GUAN
Chinese Journal of Pathology 2009;38(12):833-834
Aged
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Aged, 80 and over
;
Alzheimer Disease
;
metabolism
;
pathology
;
Brain
;
metabolism
;
Dentate Gyrus
;
metabolism
;
Dynamin I
;
metabolism
;
Hippocampus
;
metabolism
;
Humans
;
Monomeric Clathrin Assembly Proteins
;
metabolism
;
Neuropil
;
metabolism
;
Synaptophysin
;
metabolism
5.Characteristics of a novel human testis-specific gene, HSD-9, and its encoding protein.
Yong WANG ; Yi-nan CHEN ; Yuan QIAO ; Shi-ying MIAO ; Lin-fang WANG
Acta Academiae Medicinae Sinicae 2007;29(3):318-323
OBJECTIVETo study the characteristics of a novel human testis-specific gene, HSD-9, and its encoding protein.
METHODSHSD-9 was a novel gene from a human germ cells-specific ESTs library established in our laboratory. We used an electronic cloning method to obtain HSD-9 gene, and analyzed the characteristics of this novel gene and encoding product by bioinformatics methods, detected its expressing profile using a Northern blot assay, prepared specific rabbit polyclonal antibodies against HSD-9 protein, observed the localization of this protein in the germ cells and some somatic cells with confocal microscopy.
RESULTSHSD-9 was expressed in human testes, and its rat homolog was found in the varying germ cells. HSD-9 protein could partly be colocalized with clathrin.
CONCLUSIONSHSD-9 is specific in human testes, and the expression pattern of its encoding product is similar to those of some endocytosis proteins. It is speculated that HSD-9 protein may function in the endocytosis.
Amino Acid Sequence ; Animals ; Base Sequence ; Clathrin ; metabolism ; Humans ; Male ; Membrane Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Organ Specificity ; Rabbits ; Rats ; Testis ; metabolism
6.Interaction between functional nano-hydroxyapatite and cells and the underlying mechanisms.
Min YANG ; Yanzhong ZHAO ; Guohui WANG ; Juan TAN ; Shaihong ZHU
Journal of Central South University(Medical Sciences) 2016;41(9):937-945
OBJECTIVE:
To explore the interaction between arginine functionalized hydroxyapatite (HAP/Arg) nanoparticles and endothelial cells, and to investigate mechanisms for endocytosis kinetics and endocytosis.
METHODS:
Human umbilical vein endothelial cells (HUVECs) were selected as the research model.Cellular uptake of HAP/Arg nanoparticles were observed by laser scanning confocal microscopy.Average fluorescence intensity of cells after ingestion with different concentrations of HAP/Arg nanoparticles were determined by flow cytometer and atomic force microscopy.
RESULTS:
The HAP/Arg nanoparticles with doped terbium existed in cytoplasm, and most of them distributed around the nucleus area after cellular uptake by HUVECs. Cellular uptake process of HAP/Arg nanoparticles in HUVECs was in a time and concentration dependent manner. 4 h and 50 mg/L was the best condition for uptake. HAP/Arg nanoparticles were easier to be up-taken into the cells than HAP nanoparticles without arginine functionalized.
CONCLUSION
HAP/Arg nanoparticles are internalized by HUVECs cells through an active transport and energy-dependent endocytosis process, and it is up-taken by cells mainly through caveolin-mediated endocytosis, but the clathrin-dependent endocytic pathway is also involved..
Arginine
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pharmacology
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Biological Transport, Active
;
physiology
;
Caveolins
;
physiology
;
Cells, Cultured
;
Clathrin
;
physiology
;
Durapatite
;
pharmacokinetics
;
Endocytosis
;
physiology
;
Human Umbilical Vein Endothelial Cells
;
cytology
;
Humans
;
Nanoparticles
;
metabolism
8.Identification of Differentially Expressed Genes in Murine Hippocampus by Modulation of Nitric Oxide in Kainic Acid-induced Neurotoxic Animal Model.
Yo Ahn SUH ; O Min KWON ; So Young YIM ; Hee Jae LEE ; Sung Soo KIM
The Korean Journal of Physiology and Pharmacology 2007;11(4):149-154
Kainic acid (KA) causes neurodegeneration, but no consensus has been reached concerning its mechanism. Nitric oxide may be a regulator of the mechanism. We identified differentially expressed genes in the hippocampus of mice treated with kainic acid, together with or without L-NAME, a nonselective nitric oxide synthase inhibitor, using a new differential display PCR method based on annealing control primers. Eight genes were identified, including clathrin light polypeptide, TATA element modulatory factor 1, neurexin III, ND4, ATPase, H+ transporting, V1 subunit E isoform 1, and N-myc downstream regulated gene 2. Although the functions of these genes and their products remain to be determined, their identification provides insight into the molecular mechanism(s) involved in KA-induced neuronal cell death in the hippocampal CA3 area.
Animals*
;
Cell Death
;
Clathrin
;
Consensus
;
Hippocampus*
;
Kainic Acid
;
Mice
;
Models, Animal*
;
Neurons
;
NG-Nitroarginine Methyl Ester
;
Nitric Oxide Synthase
;
Nitric Oxide*
;
Polymerase Chain Reaction
;
Proton-Translocating ATPases
9.Cytomorphology and Molecular Characterization of CLTC-ALK Rearrangement in 2 Cases of ALK-Positive Diffuse Large B-cell Lymphoma with Extensive Bone Marrow Involvement.
Hee Suk CHOUNG ; Hee Jin KIM ; Won Seog KIM ; Kihyun KIM ; Sun Hee KIM
The Korean Journal of Laboratory Medicine 2008;28(2):89-94
Aanaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (DLBCL) is an unusual disease entity first reported in 1997 as DLBCL with expression of full-length ALK protein. The World Health Organization classification enlists the disease as a rare variant of DLBCL. Herein we describe two cases of ALK-positive DLBCL with cytomorphologic and molecular characteristics for the first time in Korea. The patients were 35-yr-old and 24-yr-old male patients. Immunohistochemical studies on the lymph nodes revealed large sized neoplastic cells with plasmablastic differentiation, which were negative for CD30 and positive for ALK with the characteristic granular staining in the cytoplasmic region. Extensive involvement of bone marrow was observed in both cases showing large, extremely atypical cells. Fluorescence in situ hybridization and molecular studies on the bone marrow aspirate specimens led to the detection of a clathrin (CLTC)/ALK rearrangement. Despite aggressive chemotherapy, the patients died 15 and 17 months after the diagnosis, indicating poor prognosis of the disease entity. This is the first report demonstrating the cytomorphologic findings of ALK-positive DLBCL cells on bone marrow aspirates.
Adult
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Bone Marrow/*pathology
;
Clathrin/*genetics
;
Fatal Outcome
;
*Gene Fusion
;
Humans
;
Immunohistochemistry
;
In Situ Hybridization, Fluorescence
;
Lymphoma, Large B-Cell, Diffuse/*genetics/metabolism/*pathology
;
Male
;
Protein-Tyrosine Kinases/*genetics
;
Reverse Transcriptase Polymerase Chain Reaction
;
Sequence Analysis, DNA
10.Clathrin and Lipid Raft-dependent Internalization of Porphyromonas gingivalis in Endothelial Cells.
Sang Yong KIM ; So Hee KIM ; Eun Kyoung CHOI ; Yun Woong PAEK ; In Chol KANG
International Journal of Oral Biology 2014;39(3):131-136
Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.
Actins
;
Caveolae
;
Caveolin 1
;
Clathrin*
;
Endothelial Cells*
;
GTP Phosphohydrolases
;
Human Umbilical Vein Endothelial Cells
;
Humans
;
Microtubules
;
Phosphatidylinositol 3-Kinase
;
Phosphotransferases
;
Polymerization
;
Polymers
;
Porphyromonas gingivalis*
;
RNA, Small Interfering
;
src-Family Kinases