We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.
Blotting, Northern ; Bucladesine/pharmacology* ; Bucladesine/antagonists & inhibitors ; Enzyme-Linked Immunosorbent Assay ; Fibronectins/metabolism ; Fibronectins/genetics* ; Fibrosarcoma/genetics* ; Gene Expression Regulation* ; Human ; Luciferase/metabolism ; Precipitin Tests ; Promoter Regions (Genetics) ; RNA, Messenger/metabolism ; Tetradecanoylphorbol Acetate/pharmacology* ; Transfection ; Tumor Cells, Cultured ; beta-Galactosidase/metabolism

Prediction of individual outcome after cardiopulmonary resuscitation is of major medical, ethical, and socioeconomic interest but uncertain. We experienced the case thats the patient got complete neurologic recovery after the 123th day firm cardiac arrest, who had been suspected to go with poor prognosis because she got the findings of Glasgow Coma Scale 4, severe diffuse encephalopathy on encephalogram and generalized tonic-clonic seizure at the 4th day. Recently, a 29 year-old women who sustained from respiratory arrest induced presumably by sedative and anticonvulsant therapy for control of seizure that happened during local lidocaine anesthesia far mamoplasty was transfered to our emergency medical center from local private plastic office. Arrest time was about 20 minutes. On hospital arrival, she had a pulseless bradyasystole and no respiration, but spontaneous circulation was restored at 10 minutes artier CPR started. We started cerebral oriented resuscitation including mild hypothermia(34degrees C), hemodilution, calcium channel blocker infusion. On hospital day 4, patient's glasgow coma scale(GCS) was 4. On hospital clay 7, Brain Magnetic Resonance Imaging(MRI) showed high signal intensity on T2WI, involving the bilateral basal ganglia. After contrast administraton, marked enhancement can be seen at the lesion site. Patient's glasgow coma scale(GCS) increased step by step to 5 on 8th day, 7 on 14th day, 10 on 15th day, 13 on 17th day, 15 on 20th day. 40 days later the patient was discharged with minor neurologic abnormality including hand tremor, dysphonia, amenorrhea and Mini Mental State Examination(MMSE) score(26). Long-term Follow up revealed that all neurologic functional abnormality inducting hand tremor, dysphonia, amenorrhea and MMSE score(26) is completely recovered on 123th day after episode of cardiopulmonary arrest.
Adult ; Amenorrhea ; Anesthesia ; Basal Ganglia ; Brain ; Calcium Channels ; Cardiopulmonary Resuscitation ; Coma ; Dysphonia ; Emergencies ; Female ; Follow-Up Studies ; Glasgow Coma Scale ; Hand ; Heart Arrest* ; Hemodilution ; Humans ; Hypoxia-Ischemia, Brain* ; Lidocaine ; Plastics ; Prognosis ; Respiration ; Resuscitation ; Seizures ; Tremor

In diabetes mellitus, thickening of basement membrane in capillaries and small vessels is a well-known finding and important in the progression of diabetic microangiopathy. We evaluated whether the plasma levels of type IV collagen and fibronectin, which are important factors of basement membrane, are related with the presence of diabetic microangiopathy. Plasma type IV collagen and fibronectin levels were measured in 40 healthy controls (Mean +/- SD, age; 50.3 +/- 5.5 yr) and 94 diabetic patients (age; 52.4 +/- 13.5 yr) with and without microvascular complications. The mean plasma levels of type IV collagen (5.3 +/- 2.9 ng/ml) and fibronectin (474.4 +/- 119.4 ug/ml) in diabetic patients were significantly higher (p < 0.01) than in healthy controls (3.7 +/- 1.3 ng/ml and 319 +/- 50.9 ug/ml). The mean plasma level of type IV collagen in diabetic patients with complications (6.6 +/- 3.7 ng/ml) was significantly higher (p < 0.01) than in those without complications (4.3 +/- 1.7 ng/ml) and became higher in more complicated patients. Furthermore, the severity of retinopathy and several indicators of nephropathy such as serum BUN, creatinine and proteinuria were closely associated with plasma type IV collagen level and a significant correlation was found between plasma type IV collagen and creatinine clearance (r = -0.31, p < 0.001). There was no significant difference in plasma fibronectin concentrations, however, between the diabetic patients with complications and those without complications.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult ; Aged ; Biological Markers/blood ; Blood Proteins/urine ; Blood Urea Nitrogen ; Collagen/*blood ; Creatinine/blood ; Diabetic Angiopathies/*blood/diagnosis ; Diabetic Nephropathies/blood/diagnosis ; Diabetic Retinopathy/blood/diagnosis ; Female ; Fibronectins/*blood ; Human ; Male ; Middle Age

Fibronectin, a glycoprotein, is present in plasma and extracellular matrix and is responsible for cellular adhesion. Fibronectin was purified from autologous plasma of two persistent corneal epithelial defect patients by affinity chromatogaphy and administered topically 500 micro ml, 2 dropsper time, five times a day for 3 weeks. Reepithelization began three to seven days after initiation of treatment with autologous purified fibronectin eyedrops, and epithelial defects healed nearly completely and subjective symptoms were relieved. No side effects were observed. Over a follow-up period of 4 to 23 wks, no recurrences were noted.
Extracellular Matrix ; Fibronectins* ; Follow-Up Studies ; Glycoproteins ; Humans ; Ophthalmic Solutions ; Plasma ; Recurrence

Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.
Animal ; Calcineurin/antagonists & inhibitors* ; Cell Line, Transformed ; Cell Transformation, Viral ; Cyclosporine/pharmacology* ; Enzyme Inhibitors/pharmacology ; Fibroblasts ; Fibronectins/metabolism ; Fibronectins/genetics* ; Gene Expression Regulation* ; Human ; Lung/cytology ; Mice ; Osteoblasts ; Tacrolimus/pharmacology ; Tacrolimus/analogs & derivatives*

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.
3T3 Cells ; Adipocytes/cytology/*metabolism ; Animal ; Cell Differentiation/*genetics ; Cell Line ; Collagen Type I/*genetics/metabolism ; Down-Regulation/genetics ; Gene Expression Regulation ; Genes, Reporter ; Kinetics ; Mice ; Mutation ; Procollagen/*genetics/metabolism ; Promoter Regions (Genetics) ; RNA, Messenger/metabolism ; Repressor Proteins/genetics/metabolism ; Transcription, Genetic

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.
3T3 Cells ; Adipocytes/cytology/*metabolism ; Animal ; Cell Differentiation/*genetics ; Cell Line ; Collagen Type I/*genetics/metabolism ; Down-Regulation/genetics ; Gene Expression Regulation ; Genes, Reporter ; Kinetics ; Mice ; Mutation ; Procollagen/*genetics/metabolism ; Promoter Regions (Genetics) ; RNA, Messenger/metabolism ; Repressor Proteins/genetics/metabolism ; Transcription, Genetic

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.
src-Family Kinases/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/*physiology ; Signal Transduction/*physiology ; Receptors, Vitronectin/genetics/*physiology ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Paxillin/metabolism ; Myocytes, Smooth Muscle/drug effects/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects/*metabolism ; Morpholines/pharmacology ; Molecular Sequence Data ; Integrins/genetics/*physiology ; Humans ; Flavonoids/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors ; Extracellular Matrix Proteins/genetics/*physiology ; Enzyme Inhibitors/pharmacology ; Chromones/pharmacology ; Cells, Cultured ; Cell Movement/*physiology ; Cell Adhesion/physiology ; Animals ; Amino Acid Sequence ; Amino Acid Motifs/genetics ; 1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors

OBJECTIVES: Cerebral infarction as a macrovascular complication in patients with diabetes mellitus is frequent. However, mechanisms for the development of cerebral infarction were not well known until today. The aims of this study were 1) to determine the relationship between the fibrinogen B beta 448 polymorphism and fibrinogen levels in patients with cerebral infarction, and 2) to assess usefulness of fibrinogen B beta 448 polymorphism as a marker of cerebral infarction in patients with diabetes mellitus. METHODS: We studied 60 diabetes mellitus patients, 26 diabetes mellitus patients with cerebral infarction, 17 cerebral infarction patients, and 121 normal controls. Fibrinogen B beta 448 genotype was determined by the PCR-RFLP method using restriction enzyme Mnl I. RESULTS: Fibrinogen levels in each patient group were not significantly different from one another. Fibrinogen B beta 448 genotype frequencies of the patient groups did not significantly differ from those of the normal controls. CONCLUSION: This study didn't show the relationship between the fibrinogen B beta 448 polymorphism and fibrinogen levels in patients with cerebral infarction. Moreover, these data didn't suggest the fibrinogen B beta 448 polymorphism as a marker of cerebral infarction in patients with diabetes mellitus. Further studies are needed to find the other polymorphic sites of fibrinogen gene which can affect the levels of fibrinogen.
Cerebral Infarction* ; Diabetes Mellitus* ; Diabetes Mellitus, Type 2 ; Fibrinogen* ; Genotype ; Humans

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.
Angiogenesis Inhibitors/chemistry/*pharmacology ; Animals ; Antineoplastic Agents/chemistry/*pharmacology ; Base Sequence ; Benzocaine/chemistry/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Chloramphenicol/chemistry/*pharmacology ; DNA Primers ; Drug Combinations ; Factor VIII/chemistry/*pharmacology ; Humans ; Integrin alpha5beta1/*physiology ; Integrin alphaVbeta3/*physiology ; Male ; Mice ; Mice, Inbred BALB C ; Nitrofurazone/chemistry/*pharmacology ; Recombinant Fusion Proteins/chemistry/*pharmacology