The pathogenesis of diabetic retinopathy has not been fully explained. The earliest histological lesion is the loss of intramural pericytes and thickening of the basement membrane. Increased activity of the polyol pathway is a probable mechanism for these two abnormalities. Investigations have suffered from the lack of an exact animal model simulating the human condition. Examination of the retina in the spontaneously diabetic SHR/N:Mcc-cp rat demonstrated degeneration and loss of intramural pericytes, a progressive increase in basement membrane thickness, and microinfarctions with an area of non-perfusion. Therefore, this model may be used to clarify the biochemical mechanisms linking the metabolic abnormalities of diabetes and retinopathy.
Animal ; Diabetic Retinopathy/pathology* ; Disease Models, Animal ; Female ; Hybridization ; Male ; Rats ; Rats, Inbred SHR/genetics ; Rats, Inbred Strains/genetics ; Retina/pathology* ; Retinal Degeneration/pathology

OBJECTIVETo identify differentially expressed genes related to asthma by using a rat model.

METHODSTotal RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. The white clones in selective medium from cDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank (NCBI).

RESULTSThere were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search (http://www.ncbi.hlm.nih.gov/BLAST) revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments.

CONCLUSIONThe forward-subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.

Animals ; Asthma ; genetics ; DNA, Complementary ; genetics ; Female ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Library ; Male ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; Rats ; Rats, Inbred Strains

OBJECTIVETo observe the expression of protein arginine N-methyltransferase (PRMT) genes in the lung and spleen of E3 rats with acute asthma.

METHODSE3 rats with ovalbumin-induced pulmonary inflammation were divided into two groups (n=10), and the validity of the acute asthma model was evaluated by histological observation with HE and PAS staining and by measurement of NO production. Semi-quantitative RT-PCR was employed to detect the expressions of PRMT1-PRMT6 genes in the lung and spleen tissues of the rats.

RESULTSIn the lung tissue of the asthmatic rats, the gene expressions of PRMT1 (P<0.01), PRMT2 (P<0.01), PRMT3 (P<0.05) and PRMT5 (P<0.05) were significantly increased, but the expression of PRMT4 gene (P<0.05) was significantly decreased as compared with those in the control tissue. In the spleen tissue of the asthmatic rats, the expressions of PRMT2 (P<0.05) and PRMT5 genes (P<0.05) showed a significant increase as compared with those in the control rat tissue.

CONCLUSIONThe gene expressions of PRMTs vary significantly between asthmatic rats and control rats, suggesting that PRMTs play an important role in the post-translational modification process of asthma-related genes.

Acute Disease ; Animals ; Asthma ; enzymology ; Female ; Male ; Protein Processing, Post-Translational ; Protein-Arginine N-Methyltransferases ; classification ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred Strains

OBJECTIVETo construct a subtractive cDNAs library of up-regulated genes in rat hepatic stellate cells (HSCs) stimulated with platelet-derived growth factor (PDGF)-BB by suppression subtractive hybridization (SSH) technique, to clone the up-regulated genes associated with its regulation effects, and to elucidate the mechanism of the molecular biology of hepatic fibrosis involved in PDGF-BB.

METHODSThe mRNA was isolated from HSCs stimulated with PDGF-BB and controlled with identical cells untreated with PDGF-BB, and then the cDNAs were synthesized. The cDNAs were designated as tester and driver. After being digested by restriction enzyme Rsa I, small-sized cDNAs were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, individually. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.

RESULTSThe subtractive cDNAs library of up-regulated genes in HSCs stimulated with PDGF-BB was constructed successfully. The amplified library contained 102 positive clones. Colony PCR showed that 93 clones contained 200-1000 bp inserts. Sequence analysis was performed in 31 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank; altogether 13 coding sequences were obtained, which were known ones. The genes mainly included voltage-dependent anion channel (VDAC), heat shock protein 47 and RAN-member RAS oncogene family genes.

CONCLUSIONActing as one of the most effective mitogens, PDGF-BB up-regulated some gene expressions during stimulation of the HSCs, including some cell growth associated proteins, some proteins participating in intracellular metabolism and some molecular chaperone proteins. This work brings some new clues for studying the molecular biological mechanism involved in the up-regulated genes in PDGF-BB transactivated HSCs in hepatic fibrosis.

Animals ; Gene Expression ; Gene Library ; Hepatic Stellate Cells ; Nucleic Acid Hybridization ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; genetics ; Rats ; Rats, Inbred Strains ; Up-Regulation

OBJECTIVETo research the effects and mechanism of Shenqi compound (SQC) on PTEN/ PI3K signal transducing path and angiogenesis in Goto-Kakizaki (GK) rats with diabetes mellitus type 2 (DM2) caused macroangiopathy.

METHODGK rats with blood sugar > or = 11.1 mmol/L were divided into 4 groups, the GK group, the model group, the Western medicine (WM) group treated by atorvastatin 1.5 mg/(kg x d) and the Chinese medicine (CM) group treated with SQC 1.44 g/(kg x d). All were fed 35 days with high fatty diet, but to the latter three groups, N omega-nitriyl-L-arginine methyl ester 0.1 mg/mL was added into their drinking water for macroangiopathy model establishing. Besides, a group of normal Wistar rats fed with ordinary forage was set for control. Rat's blood glucose and lipids were measured, morphology of abdominal aorta wall tissue was observed with HE staining, and mRNA expressions of PTEN and PI3Kp85 in aortic wall were detected by Real-time PCR.

RESULTSGeneral condition, gluco-lipid metabolism and aortic morphology in the CM group were significantly better than those in the model group. PTEN mRNA expression in the CM group (1.10 +/- 0.48) was significantly higher than that in the GK group (0.63 +/- 0.16) and the model group (0.17 +/- 0.07, both P < 0.01), but near to that in the WM group (1.11 +/- 0.46), while the PI3Kp85 mRNA expression in the TCM group (0.19 +/- 0.05) was lower than that in the GK group (1.38 +/- 0.43, P < 0.01), but near to that in the model group (0.33 +/- 0.09) and the WM group (0.11 +/- 0.06, both P > 0.05).

CONCLUSIONSQC could increase the PTEN mRNA expression and restrain the PI3Kp85 mRNA expression in aorta, which is possibly the partial mechanisms of action of the remedy in inhibiting angiogenesis and preventing diabetic macroangiopathy.

Angiogenesis Inhibitors ; pharmacology ; therapeutic use ; Animals ; Aorta ; metabolism ; Astragalus membranaceus ; chemistry ; Atherosclerosis ; prevention & control ; Class Ia Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; drug therapy ; metabolism ; Diabetic Angiopathies ; drug therapy ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Male ; PTEN Phosphohydrolase ; genetics ; metabolism ; Panax ; chemistry ; Phytotherapy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred Strains ; Rats, Wistar ; Signal Transduction ; drug effects

Clinical cases of type-1 hypersensitive reaction to rice (Oryza sativa) have been reported in western countries as well as in Japan. Among rice proteins, 14-16 kD globulin proteins encoded by multiple gene family have been identified as major rice allergens. In this study, a rice cDNA library was constructed using lambda UniZap vector and screened with a rat anti-16 kD globulin protein polyclonal antibody in order to isolate Korean rice allergenic cDNA clones. Five independent cDNA clones, termed RAK1-5, were obtained after second rounds of plaque assay and immunoblot analysis. These clones encoded 13-19 kD recombinant proteins upon IPTG induction, which were identified by the polyclonal antibody in immunoblot analysis. DNA sequencing analysis showed that RAK1-4 have 99% sequence homology with RA5b, and RAK5 is closely related with RA14c. This result indicated that RA5b gene is widely distributed in our cDNA library among other possible rice allergenic genes, and more study is needed to isolate heterogeneous or novel rice allergen genes. Copyright 2000 Academic Press.
Allergens/immunology ; Allergens/genetics* ; Amino Acid Sequence ; Animal ; Antibodies/immunology ; Antibody Specificity ; Cloning, Molecular ; Cross Reactions ; DNA, Complementary ; Female ; Gene Library ; Genetic Vectors ; Immunoblotting/methods* ; Molecular Sequence Data ; Plant Proteins/immunology ; Plant Proteins/genetics* ; Rats ; Rats, Inbred Strains ; Rice/genetics* ; Sequence Analysis, DNA

Ceramide, a product of sphingomyelin hydrolysis, is now recognized as an intracellular lipid messenger, which mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. Recently, ceramide has been implicated in the regulation of phospholipase D (PLD). In this study, we examined the effects of ceramide on the activity and mRNA level of PLD during apoptotic process in FRTL-5 thyroid cells. C2-ceramide (N-acetyl sphingosine) induced apoptosis in FRTL-5 thyroid cells. Fluorescent staining showed that ceramide induced the typical features of apoptosis including condensed or fragmented nuclei. DNA fragmentation was also observed by agarose gel electrophoresis. Flow cytometric cell cycle analysis showed more clearly that ceramide induced apoptotic cell death in FRTL-5 thyroid cells. The treatment of FRTL-5 thyroid cells with thyroid-stimulating hormone (TSH) resulted in an increased PLD activity in a dose- and time-dependent manner. However, the TSH-induced increase in PLD activity was down-regulated within 2 h after ceramide treatment. Furthermore, the levels of PLD mRNA were found to be decreased throughout apoptotic process as inferred by reverse transcription-polymerase chain reaction. However, the decreases in PLD mRNA levels were not correlated with those in PLD activities after ceramide treatment. Taken together, these data suggest that ceramide inhibits the PLD activity in an early apoptotic phase and down-regulation of the levels of PLD mRNA may be implicated in apoptotic process in FRTL-5 thyroid cells.
Animal ; Apoptosis/drug effects* ; Cells, Cultured ; DNA Fragmentation ; Enzyme Activation/drug effects ; Flow Cytometry ; Gene Expression Regulation, Enzymologic/drug effects ; Phospholipase D/metabolism* ; Phospholipase D/genetics ; RNA, Messenger/genetics ; Rats ; Rats, Inbred Strains ; Sphingosine/pharmacology ; Sphingosine/analogs & derivatives* ; Thyroid Gland/enzymology ; Thyroid Gland/drug effects* ; Thyrotropin/pharmacology

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.
Albumins/metabolism ; Androstadienes/*administration & dosage/pharmacology ; Animals ; Creatinine/blood ; Desmin/genetics/metabolism ; Diabetes Mellitus, Experimental/*drug therapy/metabolism/pathology ; Diabetic Nephropathies/*drug therapy/metabolism/pathology ; Disease Models, Animal ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Kidney/*pathology ; Membrane Proteins/genetics/metabolism ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Podocytes/*drug effects/metabolism/pathology ; Rats ; Rats, Inbred Strains ; cdc42 GTP-Binding Protein/genetics/metabolism ; rac1 GTP-Binding Protein/genetics/metabolism