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.
Alkaline Phosphatase/pharmacology ; Animal ; Brain/metabolism ; Calpain/metabolism ; Carrier Proteins/*chemistry ; Caspases/metabolism ; Cattle ; 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/metabolism ; Reticulocytes/metabolism ; Support, Non-U.S. Gov't ; Translation, Genetic ; src Homology Domains

This study was undertaken to investigate mineral changes in weathered scalp hair after burial. EDX (energy dispersive X-ray spectroscopy) analysis was performed to measure the presence of minerals on the hair surface. Twelve scalp hairs, buried for 5-40 years, were chosen from deceased individuals buried in tombs in Soha-Ri, Kyonggi-Do, and other regions in Korea. Three normal hairs were used as the control group. EDX data showed that carbon, oxygen, and sulfur were detected in hair collected from all three burial grounds. In contrast, calcium was only detected in hair collected from tombs in Soha-ri. The amounts of calcium and sulfur were found to decrease with time for hair collected from tombs in Soha-ri. Similar results were observed with sodium for hair collected from other regions. These results show region specific mineral detection and a decrease in the concentration of minerals with time. Consequently, it is suggested that changes in minerals concentration in weathered hair could be used as basic data in the field of forensic medicine.
Burial ; Calcium ; Carbon ; Forensic Medicine ; Forensic Sciences ; Hair ; Humans ; Korea ; Minerals ; Oxygen ; Scalp ; Sodium ; Spectrometry, X-Ray Emission ; Sulfur ; Weather

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.
Adaptor Proteins ; Animal ; Brain Chemistry ; Calpain/*metabolism ; Carrier Proteins ; Caspases/*metabolism ; Cattle ; Clathrin/*metabolism ; Coated Pits, Cell-Membrane/*metabolism ; Hydrolysis ; Membrane Proteins ; Molecular Weight ; Nerve Tissue Proteins/chemistry/*isolation & purification/metabolism ; Neurons/*chemistry ; Protein Binding ; Protein Conformation ; Recombinant Proteins/chemistry/metabolism

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.
Adaptor Proteins ; Animal ; Brain Chemistry ; Calpain/*metabolism ; Carrier Proteins ; Caspases/*metabolism ; Cattle ; Clathrin/*metabolism ; Coated Pits, Cell-Membrane/*metabolism ; Hydrolysis ; Membrane Proteins ; Molecular Weight ; Nerve Tissue Proteins/chemistry/*isolation & purification/metabolism ; Neurons/*chemistry ; Protein Binding ; Protein Conformation ; Recombinant Proteins/chemistry/metabolism

No abstract available.
Kasabach-Merritt Syndrome*

No abstract available.
Animals ; Brain* ; Cats* ; Magnetic Resonance Spectroscopy*

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

Although much is known about the molecular biology of platelets, the megakaryocytes' (MKs) molecular biology was not understood so well because of their rareness. By the cloning and characterization of thrombopoietin (TPO), which is the principal regulator of the growth and development of the MKs, researches on the MKs have been growing rapidly. To understand megakaryocytopoiesis, we investigated the gene expression profile of the MKs using oligonucleotide microarray where 10, 108 unique genes were spotted. Comparing the fluorescence intensities of which ratio is > or = |2|, 372 genes were up-regulated and 541 genes were down-regulated in MKs. For confirmatory expression, RNase protection assay (RPA) establishing abundant apoptotic gene expression was carried out. In MKs, many of the known genes, including several platelet related genes, GATA binding protein were highly expressed. Particularly, TGF beta, clusterin (complement lysis inhibitor), and thymosin beta 4 (actin-sequestering molecules) were expressed highly in MKs. As MKs specific expressed genes may regulate normal and pathologic platelet (and/or MK) functions, the transcript profiling using microarray was useful on molecular understanding of MKs.
Blood Platelets ; Carrier Proteins ; Clone Cells ; Cloning, Organism ; Clusterin ; Fluorescence ; Gene Expression* ; Growth and Development ; Humans* ; Megakaryocytes* ; Molecular Biology ; Oligonucleotide Array Sequence Analysis ; Ribonucleases ; Thrombopoiesis ; Thrombopoietin* ; Thymosin ; Transcriptome ; Umbilical Cord*

No abstract available.

PURPOSE: Study was done on the lower urinary tract symptoms(LUTS) associated with the benign prostatic hyperplasia(BPH) in the community based populations in Chungbuk province, and correlation among these symptoms, age, prostate volume, and flow rate was evaluated. MATERIALS AND METHODS: All men(764 men) living in Chungbuk province in Korea were invited to undergo measurements of peak flow rate, TRUS and to complete symptom scores and previously validated lifestyle questionnaire. RESULTS: Moderate to severe symptoms(symptom scores 8 or more) were found in 348 of our 695 samples(50.1%). A good correlation was observed between the total symptom score and quality of life questionnaire(r=0.68, P<0.001). The IPSS was weakly correlated with age(r=0.372, P<0.001) or peak flow rate(r=-0.27, P<0.001). Additional aim of this study was to determine the relative impact on the prevalence rates of the inclusion of these different parameters in the case definition of BPH. The highest overall prevalence of 27.7%(Chi-square 18.62, P<0.001) occurred using the definition that combined a prostate volume >20 cm3, and IPSS > or =8, while the lowest prevalence rate of 7.64%(Chi-square 32.03, P<0.001) occurred with the definition that combined a prostate volume >20 cm3, an IPSS > or =8, peak flow rate < or =10 ml/s, and urine volume >100 ml. CONCLUSIONS: Thus, the prevalence rates depended very much on the parameters used in case definition. Follow-up would establish the necessity of a work-up request and treatment for BPH and help determine the best clinical definition of BPH.
Chungcheongbuk-do* ; Korea ; Life Style ; Prevalence* ; Prostate ; Prostatic Hyperplasia* ; Quality of Life ; Surveys and Questionnaires ; Urinary Tract