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

AIM: To study the chemical constituents and bioactivity of the seeds of Crataegus pinnatifida. METHODS: The chemical constituents were isolated and purified by macroporous adsorptive resin D101, silica gel, and ODS column chromatography, and preparative HPLC. Their structures were elucidated on the basis of spectroscopic methods. In addition, the cytotoxic activities of compounds 1-4 were investigated on OPM2 and RPMI-8226 cells. RESULTS: Four compounds were obtained and their structures were identified as (7S, 8S)-4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3, 5-dimethoxybenzaldehyde (1), (+)-balanophonin (2), erythro-guaiacylglycerol-β-coniferyl aldehyde ether (3), buddlenol A (4). CONCLUSION Compound 1 is a novel norlignan, while compounds 1-4 exhibited marginal inhibition on the proliferation of OPM2 and RPMI-8226 cells.
Cell Line ; Cell Proliferation ; drug effects ; Crataegus ; chemistry ; Humans ; Molecular Structure ; Nerve Tissue Proteins ; chemistry ; isolation & purification ; toxicity ; Plant Extracts ; chemistry ; isolation & purification ; toxicity ; Seeds ; chemistry

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

This study is to explore the effect of ginsenoside Rb1 on the process of beta-amyloid peptide(25-35) (Abeta(25-35)) -induced hyperphosphorylation of tau protein, and on the level of cyclin-dependent kinase 5 activator, p25/p35. Western blotting and/or immunocytochemical staining were used to detect the levels of phosphorylation of tau protein at the sites of Thr205, Ser396, Ser404 in hippocampal neurons, cdk5 and p25/p35. After exposure to Abeta(25-35) (20 micromol x L(-1)) for 12 h, the levels of tau protein phosphorylation at the sites of Thr205, Ser396, Ser404 were enhanced, the level of p25 was increased, but the level of protein cdk5 was not changed markedly. Pretreatment with ginsenoside Rb1 reduced Abeta(25-35) -induced hyperphosphorylation of tau protein and decreased the lever of p25, but had no effect on cdk5. Ginsenoside Rb1 can attenuate Abeta(25-35) -induced hyperphosphorylation of tau protein through CDK5 signal pathway.
Amyloid beta-Peptides ; antagonists & inhibitors ; Animals ; Cyclin-Dependent Kinase 5 ; metabolism ; Fetus ; Ginsenosides ; isolation & purification ; pharmacology ; Hippocampus ; cytology ; Nerve Tissue Proteins ; metabolism ; Neurons ; metabolism ; Panax ; chemistry ; Phosphorylation ; drug effects ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; tau Proteins ; metabolism

OBJECTIVETo study the effects of Si-Wu-Tang on serum protein of blood deficient mice b y proteomicstechnique and study the enriching and regulating blood mechanism of Si-Wu-Tang on mocular level.

METHODThe blood deficient mice was induced by using a single dose of 3.5 Gy radiation from a 60Cogamma source, and high resolution two-dimensional polyacryamide gel electrophoresis (2-DE), computer-assisted image analysis, and mass spectrometry were used to detect regulated protein by Si-Wu-Tang.

RESULT12 lower and 4 higher protein in sera could be recovered by Si-Wu-Tang, 4 protein might be DNA-dependent protein kinase catalytic subunit, Dystrophin, KIF13A, dystonin. They play a part in DNA double-stranded break repair, recombination and modulation of transcription, transportation of mannose-6-phosphate receptor, etc.

CONCLUSIONSi-Wu-Tang can regulate serum protein in blood deficient mice, resulting in improving hematopoiesis and lessening irradiated injury.

Animals ; Autoantigens ; blood ; Carrier Proteins ; Cytoskeletal Proteins ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Dystonin ; Dystrophin ; blood ; Female ; Kinesin ; blood ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; Non-Fibrillar Collagens ; blood ; Plants, Medicinal ; chemistry ; Radiation Injuries, Experimental ; blood ; Radiation-Protective Agents ; pharmacology ; Whole-Body Irradiation

OBJECTIVEChinese jianpi herbal recipe Weichangan (WCA) could increase the survival rate of advanced gastric cancer. This study was designed to investigate the molecular mechanism of WCA in treatment of gastric cancer by cDNA array, real-time quantitative PCR and immunohistochemical technique.

METHODA human gastric adenocarcinoma cell line SGC-7901 grafted onto nude mouse was used as the animal model. The mice were divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-day period or 5-fluorouracil (5-FU) over 6-day period starting at 8th day after grafting. Control animals received saline on an identical schedule. Animals were killed 41 days after being grafted. To assess the effect of therapy tumor weight was determined by a electron balance immediately after the animals killed. SP immunohistochemical method was used to detect the expression of proliferating cell nuclear antigen (PCNA) in xenografts. For detection of apoptotic cells, apoptotic indices (AI) were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method. SP method was also used to detect the expression of cleaved Caspase-3. The expression profiles in paired WCA treated gastric cancer samples and the N. S. control samples were studied by using a cDNA array representing 14, 181 cDNA clusters. The alterations in gene expression levels were confirmed by real-time quantitative PCR. SP method was used to detect the expression of Phospho-Stat3 (Tyr705) and bcl-2.

RESULTWhen compared with controls, tumor growth was significantly inhibited by treatment with the WCA or 5-FU (P < 0.01, respectively). The average of tumor inhibitory rate in WCA group was (44.32 +/- 5.67)% and 5-FU (47.04 +/- 11.33)%. The average labeling index (LI) for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group respectively. AI of human gastric cancer xenografts in nude mice was significantly increased to (9.72 +/- 4.51)% using TUNEL method in WCA group compared with the controls (2.45 +/- 1.37)%. 5-FU group was also found a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expression ESTs among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. These 45 ESTs contains 35 cloned genes and 11 unknown ESTs. By using Real-time Quantitative PCR, the expression level of Stat3 (2(-deltadeltaCT) = 0.16) , RIPX (2(-deltadeltaCT) = 0.18), ROD1 (2(-deltadeltaCT) = 0.23) and bcl-2 (2 (-deltadeltaCT) = 0.10) was lower in WCA group than that in control group respectively. The expression of Phospho-Stat3 (Tyr705) and bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively.

CONCLUSIONChinese jianpi herbal recipe WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Stat3, RIPX, ROD1 and bcl-2 gene.

Adenocarcinoma ; drug therapy ; genetics ; pathology ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Expressed Sequence Tags ; Fluorouracil ; pharmacology ; therapeutic use ; Gene Expression Profiling ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nerve Tissue Proteins ; metabolism ; Plants, Medicinal ; chemistry ; Polypyrimidine Tract-Binding Protein ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA-Binding Proteins ; metabolism ; STAT3 Transcription Factor ; metabolism ; Stomach Neoplasms ; drug therapy ; genetics ; pathology ; Xenograft Model Antitumor Assays ; methods