ABSTRACT
Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.
Subject(s)
Animals , Rabbits , Leukemia, Myeloid, Acute/genetics , CCAAT-Enhancer-Binding Protein-alpha/genetics , Haploinsufficiency/genetics , Hematopoiesis/genetics , Phenotype , Transcription Factors/genetics , Translocation, Genetic/genetics , Mice, Transgenic , Acute Disease , Flow Cytometry , GenotypeABSTRACT
This study used topographic near-infrared spectroscopy (NIRS) to characterize frontal lobe activation while individuals actively or passively listened to exciting or calm music. Participants were 22 healthy female volunteers (mean age, 21± 4.1 years). Initial analysis showed that oxy-Hb significantly decreased in many channels when subjects listened to calm music. In contrast, oxy-Hb significantly increased when subjects listened to lively music. In addition, after listening to calm music, cortisol, α-amylase, and immunoglobulin A significantly decreased. A subsequent analysis showed that oxy-Hb signif-icantly increased when subjects listened to music while clapping to its rhythm compared with when subjects listened to music only. Our study suggests that calm music reduces levels of human stress and enables effective relaxation. In addition, our data suggests that clapping to the rhythm of music increases brain activation. Therefore, active music therapy may be more effective than passive music therapy with respect to brain activation. Such active therapy (i.e., playing rather than listening to music) may improve functional and psychological status, and cooperative abilities.
ABSTRACT
Stroke remains the third leading cause of death and of adult disability worldwide. Vascular occlusion, followed by ischemic cascade, leads to irreversible tissue injury. Recombinant tissue plasminogen activator is the only FDA approved drug for the current treatment of acute ischemic stroke. However, traditional Chinese medicine has a long history and rich clinical experience in the treatment and rehabilitation of ischemic stroke. Using a classical middle cerebral artery occlusion (MCAO) stroke model, we tested the effectiveness of Yiqihuoxue calm wind (YCW) capsule on neurological function, gross pathology and oxidative stress status in MCAO rats. YCW capsule (3.36 and 6.72 g·kg of crude drug) could significantly lower Longa's score and superoxide dismutase (SOD) level, together with less necrotic cells and infarcted area. In addition to elevated MDA and downregulated iNOS expression, YCW capsule exhibited its neuroprotective effects via free radical scavenging and NO inhibition.
Subject(s)
Animals , Humans , Male , Rats , Brain Ischemia , Drug Therapy , Genetics , Metabolism , Malondialdehyde , Metabolism , Neuroprotective Agents , Oxidative Stress , Rats, Sprague-Dawley , Stroke , Drug Therapy , Genetics , Metabolism , Superoxide Dismutase , MetabolismABSTRACT
The translocation t(10;11)(p13;q14q21) has been found to be recurrent in acute lymphoblastic and myeloid leukemias, and results in the fusion of the clathrin assembly lymphoid myeloid leukemia (CALM) gene with the AF10 gene; these genes are present on chromosomes 11 and 10, respectively. Because the CALM-AF10 rearrangement is a rare chromosomal abnormality, it is not included in routine molecular tests for acute leukemia. Here, we describe the cases of 2 patients with the CALM-AF10 fusion gene. The first patient (case 1) was diagnosed with T-cell ALL, and the second patient (case 2) was diagnosed with AML. Both patient samples showed expression of the homeobox A gene cluster and the histone methyltransferase hDOT1L, which suggests that they mediate leukemic transformation in CALM-AF10-positive and mixed-lineage leukemia-AF10-positive leukemias. Both patients achieved complete remission after induction chemotherapy. The first patient (case 1) relapsed after double-unit cord blood transplantation; there was no evidence of relapse in the second patient (case 2) after allogenic peripheral blood stem cell transplantation. Since CALM-AF10- positive leukemias have been shown to have poor prognosis with conventional therapy, molecular tests for CALM-AF10 rearrangement would be necessary to detect minimal residual disease during follow-up.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Bone Marrow/pathology , Chromosomes, Human, Pair 10 , Chromosomes, Human, Pair 11 , Cord Blood Stem Cell Transplantation , Histone-Lysine N-Methyltransferase/genetics , Homeodomain Proteins/genetics , Leukemia, Myeloid, Acute/diagnosis , Monomeric Clathrin Assembly Proteins/genetics , Oncogene Proteins, Fusion/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Recurrence , Transcription Factors/genetics , Translocation, GeneticABSTRACT
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.
Subject(s)
Cattle , Alkaline Phosphatase/pharmacology , Animals , Brain/metabolism , Calpain/metabolism , Carrier Proteins/chemistry , Caspases/metabolism , Cell-Free System , Clathrin/chemistry , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Lipids/chemistry , Membrane Proteins/chemistry , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins/chemistry , Reticulocytes/metabolism , Protein Biosynthesis , src Homology DomainsABSTRACT
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Subject(s)
Cattle , Adaptor Proteins, Vesicular Transport , Animals , Brain Chemistry , Calpain/metabolism , Carrier Proteins , Caspases/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Hydrolysis , Membrane Proteins , Molecular Weight , Nerve Tissue Proteins/chemistry , Neurons/chemistry , Protein Binding , Protein Conformation , Recombinant Proteins/chemistryABSTRACT
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Subject(s)
Cattle , Adaptor Proteins, Vesicular Transport , Animals , Brain Chemistry , Calpain/metabolism , Carrier Proteins , Caspases/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Hydrolysis , Membrane Proteins , Molecular Weight , Nerve Tissue Proteins/chemistry , Neurons/chemistry , Protein Binding , Protein Conformation , Recombinant Proteins/chemistryABSTRACT
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.
Subject(s)
Rats , Animals , Clathrin/metabolism , Clathrin-Coated Vesicles/chemistry , Liver/chemistry , Nerve Tissue Proteins/isolation & purification , Phosphoproteins/isolation & purificationABSTRACT
Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.
Subject(s)
Female , Mice , Rabbits , Animals , Antibodies, Monoclonal , Calpain/chemistry , Caspases/chemistry , Clathrin-Coated Vesicles/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Escherichia coli/genetics , Glutathione Transferase/genetics , Mice, Inbred BALB C , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/chemistry , Phosphoproteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/chemistry , Protein Binding , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/chemistry , src Homology DomainsABSTRACT
Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicle (CCV) in neurons. The clathrin assembly protein gene (rCALM) was cloned from rat brain cDNA library. rCALM deduced 69 kD molecule has overall 73% amino acid homology compared with that of AP180 protein. The N-terminal domain, where amino acid sequences are very similar with AP180, harbours binding sites for clathrin and inositides, as well as possible phosphorylation sites, but the proline rich C-terminal domain is different from that of AP180. The mRNA expression of rCALM and AP180 by in situ hybridization histochemistry revealed that the rCALM mRNA was more intensely expressed than that of AP180, and the distribution patterns were different from each other. These results suggest that the rCALM mediates the assembly of clathrin in neural and supporting cells of brain, and regulates the clathrin coated-vesicle formation through phosphorylation and inositide metabolism. Copyright 2000 Academic Press.