BACKGROUND: Purified major allergens of house dust mite are essential for evaluation of the allergic mechanism in molecular basis and development of new modalities of immunemodulation. OBJECTIVE: In this study, we aimed to purif group 1 and group 2 allergens from Dermatophagoides pteronyssinus (Dp). In addition, cDNAs corresponding to Der pI and II in Korean Dp were isolated and recombinant Der p1 and Der pII were synthesized. MATERIALS AND and METHOD: Der pI allergen was purified by ammonium sulfate precipitation, anion -exchange column chromatography, and gel filtrat,ion chromatography. Der pII allergen was purified by anion exchange chromatography, gel filtration chromatography, and a preparative isoelectric focusing method. RESULTS: Eight hundred ug of Der pI and 50 ug of Der pII were obtained from 100 g of culture medium and 1 g of mite bodies, respectively. The purities of these allergens were confirmed by SDS PAGE and the strong reactivity to the patient sera was identified. In order to produce a recombinant allergens, poly(A) RNA from house dust mites were isolated and used for cDNA synthesis by RT PCR. The cDNA was inserted into prokaryotic expression vector and the vectors were transformed into E. coli. A little amount of recombinant Der pI protein was produced due to the low solubility, and 1.2 mg of recombinant Der pII was produced from 1 L of E. coli culture medium. The antigenicity of Der pI was relatively weak, however, Der pII showed a strong antigenicity. Amino acid sequence of the amplified cDNA deduced from DNA sequences of Der pII showed 6 different variants. The variation of amino acid sequences suggests the possibility of high incidence of mutation of Der pII protein. CONCLUSION: A simplified method for the purification of Der pI and Der pII was developed. Recombinant allergens will be useful for the diagnosis and treatment of allergy with lower costs.
Allergens* ; Amino Acid Sequence ; Ammonium Sulfate ; Base Sequence ; Chromatography ; Chromatography, Gel ; Dermatophagoides pteronyssinus* ; Diagnosis ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Humans ; Hypersensitivity ; Incidence ; Isoelectric Focusing ; Mites ; Polymerase Chain Reaction ; Pyroglyphidae* ; RNA, Messenger ; Solubility

Incidence of aseptic loosening of hip prostheses is increasing in recent years. Previous studies suggested the involvement of proteinases and cytokines in the accelerated bone lysis associated with loosening. To investigate the role of matrix metalloproteinases (MMPs) in the loosening, Gelatin/Type IV collagenases, namely, 72 KDa matrix metalloproteinase (MMP)-2 type and 92 KDa MMP-9 type were analyzed in 14 cases of the loosened endoprostheses of the hip. Zymographic and densitometric analyses revealed production of MMP-2 ancl elevated induction of MMP-9 in tissue extracts from both the interface hetween hone and implants and the capsular tissues when compared with those in synovium obtained from a patient with a t'ractured femoral neck. MMP-9 showed stronger activity than MMP-2. In the sample of a fractured femoral neck, MMP-2 was detected, but MMP-9 was not detected. In matched samples, the activity of MMP-2 and MMP-9 in the interface tissues showed stronger activity than those in the capsular tissues. There was no difference between cemented and uncemented femoral prostheses. The state of prostheses(loosening, osteolysis, and cup wear) did not influence on the activity of MMP-2 and MMP-9. Theses findings suggest a role for MMP-2 and MMP-9 type gelatinase/Type IV collagenases in the degradation of extracellular matrix of periprosthetic tissues, where they may cause weakening of the connective tissue hed and the loosening of total hip replacement endoprostheses. Consequently. we could confirm the role of MMP cascade in aseptic loosening of total hip prostheses. The further study ahout other types of MMP and the inhihitor of MMP will be needed.
Arthroplasty, Replacement, Hip ; Collagenases ; Connective Tissue ; Cytokines ; Extracellular Matrix ; Femur Neck ; Hip Prosthesis* ; Hip* ; Humans ; Incidence ; Matrix Metalloproteinases* ; Osteolysis ; Peptide Hydrolases ; Prostheses and Implants ; Synovial Membrane ; Tissue Extracts

The regulation of fatty acid synthase in rat liver was investigated at transcriptional and post-transcriptional levels. When rats were fasted for 3 days and refed on a high-carbohydrate diet, the amounts of FAS in liver cytosol began to increase at 12 hours and further increased until 48 hours. The amount of mRNA for FAS began to increase at 6 hours and reached to a maximum level at 12 hours, indicating that the expression of mRNA for FAS precedes the increase of FAS protein pool. After 12 hours the amounts of mRNA gradually decreased and remained at a much lowered level between 24 and 48 hours. The elevated amount of FAS mRNA reflected on the amount of FAS protein in the first 24 hours, but these two parameters were not paralleled thereafter, probably due to the changes in the translational efficiencies. The run-on transcriptional activity of FAS gene began to increase at 4 hours after refeeding a high-carbohydrate diet and further increased to reach a maximum level 25 fold of the initial level at 12 hours, followed by a 16 fold level between 24 and 48 hours. The elevation of run-on transcriptional activity of FAS gene preceded the increase of FAS mRNA in the liver cytosol by 2 hours, and a similar increasing pattern was observed until 12 hours. However, FAS mRNA concentration decreased gradually after 12 hours, while the transcriptional activity remained at a high level until 48 hours. The changes in FAS mRNA content in the cytosol of rat liver were closely related to the transcriptional activity of FAS gene in the early phase of induction, but another regulatory mechanism seems to operate in the decrease of mRNA after 12 hours.
Animal ; DNA/genetics ; Dietary Carbohydrates/administration & dosage ; Fatty Acid Synthetase Complex/*biosynthesis/genetics ; *Gene Expression Regulation, Enzymologic ; Liver/*enzymology ; Male ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; *Transcription, Genetic

The effects of insulin on ATP-citrate lyase, its mRNA in cytosol, and the transcriptional activity in nuclei of diabetic rat liver were studied. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin, and livers were removed from rats at 0, 1, 3, 6, 16, and 72 hours after the administration of insulin. ATP-citrate lyase began to increase at 16 hours, and continuously increased until 72 hours. The amount of mRNA encoding ATP-citrate lyase increased abruptly at 16 hours, then decreased to near basal level in 72 hours. No change in the transcription rate was observed until 3 hours after insulin administration. However, the activity increased 4-fold at 6 hours and 7-fold at 16 hours, 16-fold at 6 hours and 28-fold at 16 hours when pGACL1 and pGACL2 were used as probes, respectively, preceding the increase in the amounts of mRNA and the enzyme. It is suggested that the increase in the amount of ATP-citrate lyase by insulin is primarily due to the increase in the transcriptional activity of the gene in nuclei, which results in the subsequent increase in the amount of mRNA for the biosynthesis of ATP-citrate lyase in cytosol.
ATP Citrate (pro-S)-Lyase/*biosynthesis/genetics ; Animal ; Cell Nucleus/enzymology ; Cytosol/enzymology ; Diabetes Mellitus, Experimental/*enzymology ; Enzyme Induction/drug effects ; Insulin, Isophane/*pharmacology ; Liver/*enzymology ; Male ; RNA, Messenger/drug effects ; Rats ; Rats, Sprague-Dawley ; Transcription, Genetic/drug effects

It has been suggested that glucose metabolites and insulin are the most important factors inducing ATP-citrate lyase (ACL) by a high carbohydrate diet. We have used a primary culture of rat hepatocytes to confirm the role of glucose and insulin in terms of ACL gene expression. The results showed that glucose displayed a direct effect on ACL gene expression and the insulin helps the glucose effect. The nucleotide sequences from -512 to -485 of the ACL promoter are highly homologous (70%) to the sequences surrounding the carbohydrate response element (ChoRE) of the S14 gene. The gel retardation analysis using ChoRE of the S14 gene showed that the ACL promoter which contains the ChoRE-like sequence specifically inhibited the formation of the complex by the nuclear proteins isolated from rat liver. To localize the regions which are involved in the regulation of ACL gene expression, transient expression assay using ACL promoter-CAT (chloramphenicol acetyltransferase) constructs containing various lengths of a 5' flanking region of the ACL gene were carried out. The proximal promoter region -419 to -1 containing several potential Sp1 binding sites showed the strong enhancing effect, which increases the transcription of CAT genes in the various cell lines, such as the CHO (Chinese hamster ovary) cell, the HepG2 cell, and primary cultured rat hepatocytes. In response to glucose, among the ACL promoter-CAT constructs, only pNP33-CAT (-1342 to -1) showed a 2.64 fold increase in CAT activity by a high concentration of glucose. The activation of ACL gene expression by glucose seems to be regulated in a complicated manner involving interactions between the contexts of the several sequence elements and various transacting factors, which is not a simple mechanism directed only by a short sequence element.
ATP Citrate (pro-S)-Lyase/*genetics ; Animal ; Base Sequence ; CHO Cells ; Cells, Cultured ; Female ; *Gene Expression Regulation, Enzymologic ; Glucose/pharmacology ; Hamsters ; Liver/cytology/enzymology ; Molecular Sequence Data ; Promoter Regions (Genetics) ; Rats ; Support, Non-U.S. Gov't ; Transcription, Genetic

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.
ATP Citrate (pro-S)-Lyase/metabolism ; ATP Citrate (pro-S)-Lyase/genetics* ; Binding Sites ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/genetics ; DNA Footprinting/methods ; Deoxyribonuclease I/metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Enzymologic* ; Glucose/pharmacology ; Glucose/metabolism* ; Human ; Immunoblotting ; Promoter Regions (Genetics)* ; Transcription Factor, Sp1/metabolism* ; Transcription, Genetic* ; Transfection

PURPOSE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR. MATERIALS AND METHODS: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo. RESULTS: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice. CONCLUSION: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.
Animals ; Cholesterol/*blood ; Cholesterol, LDL/blood ; Hep G2 Cells ; Humans ; Mice ; Mice, Knockout ; Proprotein Convertases/*metabolism ; Receptors, LDL/*metabolism ; Serine Endopeptidases/*metabolism ; *Small Molecule Libraries