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

OBJECTIVETo express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).

METHODSThe 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.

RESULTSThe prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.

CONCLUSIONThe fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

Animals ; Antibodies ; immunology ; isolation & purification ; Antibody Specificity ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; immunology ; Membrane Proteins ; biosynthesis ; genetics ; immunology ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

The aim of this study was to investigate the influence of neonatal isolation stress on hyperlocomotion in complexin II knockout mouse (Cplx2(-/-)). The mice were randomly divided into 4 groups: Cplx2(-/-) with stress, Cplx2(+/+) with stress, Cplx2(-/-) without stress and Cplx2(+/+) without stress. Isolation stress was employed on the pups of stress groups from the 2nd day after the postnatal to the 21st day. The PCR was used to determine the gene type and the hyperlocomotion test was employed to detect the change of animal behavior after methamphetamine or saline injection (i.p.). The results showed that the animals of all groups increased their movement after injection of 0.2 mg/kg methamphetamine in different levels (P < 0.01), compared with those injected with saline. The Cplx2(-/-) mouse with stress revealed a significant increase in the distance of free movement after injection of 0.2 mg/kg methamphetamine compared with the knockout mouse without stress (P < 0.001). When Cplx2(-/-) mouse with stress was compared with wild type with stress, Cplx2(-/-) mouse with stress had more movement (P < 0.001), indicating that Cplx2 has effect on the hyperlocomotion as well. These results suggest an involvement of stress and Cplx2 in the movement behavior of mice.
Adaptor Proteins, Vesicular Transport ; genetics ; Animals ; Animals, Newborn ; Behavior, Animal ; physiology ; Locomotion ; physiology ; Methamphetamine ; pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Mutant Strains ; Nerve Tissue Proteins ; genetics ; Social Isolation ; Stress, Psychological ; psychology

OBJECTIVETo prokaryoticly express and purify HuD protein and its RNA recognition motifs.

METHODSHuD protein was prokaryoticly expressed and purified by molecular cloning technology. Its biologic activity was testified by Western Blot.

RESULTSPurified HuD protein and its RNA recognized motifs were observed.

CONCLUSIONSThe result might aid for basic research and clinical application.

Antibodies, Antinuclear ; biosynthesis ; genetics ; isolation & purification ; Carcinoma, Small Cell ; genetics ; immunology ; metabolism ; Cloning, Molecular ; DNA, Complementary ; genetics ; ELAV Proteins ; ELAV-Like Protein 4 ; Humans ; Lung Neoplasms ; genetics ; immunology ; metabolism ; Nerve Tissue Proteins ; biosynthesis ; genetics ; isolation & purification ; Neurons ; immunology ; Paraneoplastic Syndromes, Nervous System ; genetics ; immunology ; metabolism ; RNA-Binding Proteins ; biosynthesis ; genetics ; isolation & purification

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

OBJECTIVETo isolate, identify and analyze the sex-determining gene Transformer 2 (Aaetra2) of the major vector mosquito Aedes aegypti.

METHODStBLASTn program, RT-PCR and RACE methods were used to obtain the full-length cDNA of Aaetra2. Multiple alignments of nucleotide and amino acid sequences were conducted, and the different domains in tra2 protein were indentified. RT-PCR of the total RNA extracted from different tissue from the mosquitoes in different developmental stages was performed using specific primers.

RESULTSTwo genes, namely Aaetra2-α and Aaetra2-β, were identified in different supercontig locations. The multi-transcripts were expressed by means of alternative promoters or terminators. The different domains in tra2 protein were defined as RS-rich N-terminal region, RNA recognition motif-RRM, linker region, and RS-rich C-terminal region. Both Aaetra2-α and Aaetra2-β showed sustained expression throughout the developmental stages of Ae.aegypti, and in all the tissues without a sex specificity.

CONCLUSIONAaetra2 gene has multiple isoforms and is mapped to multiple locations in the genome. Aaetra2 has conservative functional domains of the sex-determining gene tra2. For Ae.agypti, Aaetra2 shows the potential as a new target for release of insects carrying a dominant lethal (RIDL) technology based on transgenic mosquitoes.

Aedes ; genetics ; growth & development ; Amino Acid Sequence ; Animals ; Drosophila Proteins ; genetics ; Gene Expression Regulation, Developmental ; Genes, Insect ; Insect Proteins ; genetics ; isolation & purification ; Nerve Tissue Proteins ; genetics ; Phylogeny ; RNA-Binding Proteins ; genetics ; Ribonucleoproteins ; genetics ; Sequence Alignment ; Serine-Arginine Splicing Factors ; Sex Differentiation ; genetics

OBJECTIVETo obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development.

METHODSThe nestin cDNA was cloned from human neural stem cells by RT-PCR and ligated to prokaryotic expression plasmid pQE30 for construction of the recombinant vector pQE30-nestin. After sequencing, the recombinant vector was transformed into E.coli M15 and His-tagged nestin was induced by IPTG. The nestin was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blotting. BALB/c mice were immunized with the purified recombinant protein to prepare the antiserum, which was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.

RESULTSThe nestin gene was successfully cloned from human neural stem cells, which was identical to that reported in GenBank. After IPTG induction, the E.coli transformed with pQE30-nestin plasmid expressed a 25,000 His-tagged protein, which was successfully purified and identified as nestin by Western blotting. Western blotting, ELISA and immunohistochemistry demonstrated that the antiserum could specifically bind to the recombinant nestin as well as to nestin in fetal human and rat brains.

CONCLUSIONWe successfully cloned the nestin gene and expressed the nestin, and nestin mAb prepared can specifically recognize not only the recombinant nestin, but also nestin from human and rats brain tissues.

Adult Stem Cells ; cytology ; metabolism ; Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immune Sera ; immunology ; Immunohistochemistry ; Intermediate Filament Proteins ; biosynthesis ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Nervous System ; cytology ; metabolism ; Nestin ; Recombinant Proteins ; biosynthesis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction

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