BACKGROUND: Protein kinase C (PKC)s consist of three groups of isoenzyme; conventional, novel and atypical PKCs. Diacylglycerol (DAG) activates both conventional and novel PKCs, but not atypical PKCs. High glucose-induced fibronection production was shown to be mediated by activation of DAG-sensitive PKCs. In this study, we investigated whether PKC mediates IL-1beta-induced fibronectin mRNA expression, and the subtypes of PKC involved in the process. METHODS: Fibronectin mRNA level and phosphorylated PKC zeta/iota in total cell lysate were measured by Northern blot and Western blot, respectively. RESULTS: Pretreatment of HPMCs with calphostin C, a pan-PKC inhibitor, at doses of 500, 750 and 1, 000 nM caused dose-dependent inhibition of IL- 1beta (1 ng/mL)-induced fibronectin mRNA level. GF109203X, another pan-PKC inhibitor, at doses of 1, 5 and 10 microM also downregulated IL-1beta (1 ng/ mL)-induced fibronectin mRNA level in a dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKCs, stimulated fibronectin mRNA level at doses of 1, 10 and 100 nM. After prolonged treatment of the cells for 72 hr with PMA, another dose of PMA did not increase fibronectin mRNA level, while IL-1beta (1 ng/mL) still stimulated it. Pretreatment of the cells with 5, 10, 15 and 20 microM of myristoylated PKC zeta/iota pseudosubstrate inhibited IL-1beta (1 ng/mL)-induced fibronectin mRNA level in a dose-dependent manner, while 20 microM of myristoylated PKC [19-27] pseudosubstrate, given as a control, had no effect. Stimulation of fibronectin mRNA level by IL-1beta (1 ng/mL) was completely prevented by 20 microM of my ristoylated PKC zeta/iotapseudosubstrate. IL-1beta (1 ng/ mL) increased phosphorylated PKC zeta/iota, an active form of the enzyme. CONCLUSION: IL-1beta-induced fibronectin production in HPMCs occurs by way of activation of atypical PKCs (PKC zeta/iota).
Blotting, Northern ; Blotting, Western ; Fibronectins* ; Humans* ; Interleukin-1beta* ; Protein Kinase C* ; Protein Kinases* ; RNA, Messenger

PURPOSE: The expression pattern of c-jun by ionizing radiation according to cell growth state (exponential growth phase vs. stationary phase) and its relationship with cell cycle redistribution were investigated. MATERIALS AND METHODS: The exponential growth phase (day 4) and stationary phase (day 9) cells were determined from cell growth curve according to the elapse of days in CaSki. The cells were irradiated using 6 MV X-ray with a dose of 2 Gy at a fixed dose rate of 3 Gy/min. Northern blot analysis was performed with total cellular RNA and cell cycle distribution was analyzed using flow cytometry according to time-course after irradiation. RESULTS: The maximum expression of c-jun occurred 1 hour after irradiation in both exponential growth and stationary phase cells. After then c-jun expression was elevated upto 6 hours in exponential growth phase cells, but the level decreased in stationary phase cells. Movements of cells from G0-G1 to S, G2-M phase after irradiation were higher in exponential growth phase than stationary phase. CONCLUSION: c-jun may be involved in the regulation of cellular proliferation according to the growth states after irradiation.
Blotting, Northern ; Cell Cycle ; Cell Line* ; Cell Proliferation ; Flow Cytometry ; Radiation, Ionizing ; RNA

STUDY DESIGN: Evaluation of phospholipase A2 production according to cell type of human intervertebral disc. SUMMARY OF LITERATURE REVIEW: It was reported that the phospholipase A2 activity in human lumbar disc herniation was more active than that in other tissues. OBJECTIVES: The purpose of this study was to evaluate the differences between the cells of anulus fibrosus and nucleus pulposus when lactate was added to the culture medium. MATERIALS AND METHODS: Cells from the anulus fibrosus and nucleus pulposus of a human intervertebral disc were prepared enzymatically. After the monolayer was set up, the cells were divided to three groups and lactate doses of a 0mM, 2mM or 5mM were added respectively. At two week after lactate addition the production of phospholipase A2 was measured by Northern blotting. RESULTS: Cells of nucleus pulposus produced a small amount of phospholipase A2. Those of anulus fibrosus showed a high activity of phospholipase A2 production. The concentration of lactate did not influenced on the production of phospholipase A2. CONCLUSION: The anulus fibrosus has an important role in the production of phospholipase A2 and is thought to be related with generation of discogenic pain.
Blotting, Northern ; Cell Culture Techniques ; Humans* ; Intervertebral Disc* ; Lactic Acid ; Phospholipases A2* ; Phospholipases*

OBJECTIVE: To identify and localize the Placenta Growth Factor (PlGF) mRNA in term placentas and to determine whether there are any differences in the expression of PlGF mRNA in the placentas between normal and preeclamptic pregnancy. METHODS: Northern blot and In situ hybridization with 5 cases of preeclamptic placentas and 8 cases of normal controls RESULTS: In Northern blot analysis and In situ hybridization, PlGF2 mRNA (1.7 kb) band was identified and showed significant decrease in density in preeclamptic placentas compared to that of normal controls. PlGF mRNA was mainly expressed in the syncytial membrane of villous trophoblast, and in the perivascular tissue surrounding the vasculature of stem villi. CONCLUSION: Decreased levels of PlGF mRNA in preeclamptic placentas could have some unfavorable influences on trophoblast and endothelial function, thereby may play a role to the pathogenesis of preeclampsia.
Blotting, Northern ; In Situ Hybridization ; Membranes ; Placenta* ; Pre-Eclampsia ; Pregnancy ; RNA, Messenger* ; Trophoblasts

OBJECTIVE: To investigated whether lowering oxygen tension affects invasion of cultured trophoblast. METHODS: Trophoblasts were isolated from the normal placenta in early pregnancy(6-10 weeks in gestation). Isolated trophoblasts were cultured under normoxic(5% CO2, 95% humid air in incubator) and hypoxic(MERCK, 1% O2, 99% CO2) conditions for 24, 48 and 72 hours, respectively. The proliferation ability was measured using [H3] thymidine assay. Total RNA was extracted from the cultured trophoblasts. The expressions of matrix metalloproteinase(MMP-2) and tissue inhibitor of metallo- proteinase(TIMP-2) were determined by reverse transcription- polymerase chain reaction(RT-PCR) and Northern blot analysis. The invasiveness of cultured trophoblast was observed using in vitro invasion assay. RESULTS: [H] thymidine assay indicated that cellular DNA synthesis was not affected by the culture condition. The expression of MMP-2 mRNA was decreased at 24 hours and then progressively increased in the time-dependent manner in each culture condition. The expression of TIMP-2 was decreased in the time-dependent manner under hypoxic condition. In vitro invasion assay revealed that the cultured trophoblasts under hypoxic condition has more invasive ability than them under normoxic condition. CONCLUSION: These data suggests that hypoxic condition may stimulates the invasion of trophoblast in the human placentation. And MMP-2 and TIMP-2 may be related to control their invasiveness under hypoxic condition.
Blotting, Northern ; DNA ; Humans ; Oxygen ; Placenta ; Placentation ; RNA ; RNA, Messenger ; Thymidine ; Tissue Inhibitor of Metalloproteinase-2 ; Trophoblasts*

The present study was aimed at investigating the molecular regulation of the renin- angiotensin system (RAS) in two-kidney, one clip (2K1C) hypertension. The expression of renin, angiotensinogen and angiotensin II receptor genes was determined by Northern blot analysis in rats made 2K1C hypertensive for 2 or 4 weeks. The expression of renin gene was increased in the clipped kidney and decreased in the contralateral non-clipped kidney at weeks 2 and 4. The expression of angiotensinogen gene was not significantly altered at week 2, but increased at week 4 in the clipped kidney. However, it was not significantly altered in the contralateral kidney either at week 2 or 4. Nor was the expression of angiotensinogen gene significantly altered in the liver either at week 2 or 4. On the other hand, the expression of angiotensin II receptor gene was decreased at week 2, and increased at week 4 in the clipped kidney, whereas it was not significantly changed in the contralateral kidney either at week 2 or 4. In the liver, the expression of angiotensin II receptor gene was not significantly altered at week 2, but decreased at week 4. These results suggest that the components of RAS are transcriptionally regulated in 2K1C hypertension in a manner dependent on tissues and duration of hypertension.
Angiotensin II* ; Angiotensinogen* ; Angiotensins* ; Animals ; Blotting, Northern ; Hand ; Hypertension ; Kidney ; Liver ; Rats* ; Receptors, Angiotensin* ; Renin

Glutathione(GSH) has a very important role in detoxification of cells and is closely related to antitumor drug-resistance of cancer cells. In order to evaluate the importance of glutathione metabolism in the drug-resistant cancer cells, the concentration of celluar GSH and activities of y-glutamylcysteine synthetase(GCS), y-glutamyl transpeptidase (GGT) and glutathione S-transferases(GST) in the adriamycin, vincristine, or cisplatin resistant L1210 (L1210AdR; L1210VcR, or L12100s) sublines were measured. Expression and amplification of GCS, GGT, and GST-i7 genes were also observed in the parent L1210 and the drug-resistant L1210 sublines. The concentration of GSH was increased 5.34 fold in L12100s, 2.83 fold in L1210VcR, and 1.78 fo-d in L1210AdR, compared to L1210. The activities of GCS and GGT were -increased in drug-resistant L1210 sublines. The GST activity was increased in L1210VcR and L1210Cis but decreased in L1210AdR compared to L1210. Expression of GCS, GGT, and GST-rr genes were increased in the resistant L1210 sublines compare to the parent L1210 in northern blot analyses. Overexpression of GCS, GGT, and GST-77 were observed in the resistant sublines, and the increases of the concentration of glutathione and the activities of GCS and GGT in the resistant sublines may be involved in a part of the drug-resistance in the resistant sublines.
Blotting, Northern ; Cisplatin ; Doxorubicin ; Drug Resistance ; Glutathione* ; Humans ; Metabolism* ; Parents ; Vincristine

PURPOSE: We evaluated the clinical significance of the tumor growth factor, midkine (MK), in paired gastric cancer and normal tissues. MATERIALS AND METHODS: Twenty paired normal and cancer tissues were tested for MK mRNA expression by Northern blot analysis. Vessel staining was done by immunohistochemical staining using CD-31 monoclonal antibody (Dako). RESULTS: MK mRNA was mainly expressed in cancer tissues (11 versus 1). Lymph node metastasis, pathological stage and tumor differentiation did not correlate with MK expression. However, MK expression rate increased with increment in tumor size (p=0.05). Microvascular density did not correlate with tumor invasion, lymph node metastasis, and pathological stages. However, there was a tendency of vascular density increment with MK expression in T1-T2 stage. CONCLUSION: MK was mainly expressed in larger gastric cancer tissues suggesting its role in cancer growth in vivo. But no definite correlation between MK expression and tumor microvascular density was found.
Blotting, Northern ; Gene Expression* ; Lymph Nodes ; Neoplasm Metastasis ; RNA, Messenger ; Stomach Neoplasms*

Deer antler has been widely prescribed in Chinese and Korean pharmacology. Although there have been several reports concerning the effects of deer antler, such as anti-aging action, anti-inflammatory activity, antifungal action and regulatory activity of the level of glucose, the effect on bone has not determined yet. The purpose of this study was to examine the effect of deer antler on osteoblast differentiation. Hexane extract(CNH) and chloroform extract(CN-C) were acquired from deer antler(Cervus nippon) and MC3T3-E1 preosteoblasts were cultured in the presence or absence of each extract. Osteoblast differentiation was estimated with the formation of mineralized nodules and the mRNA expression of alkaline phosphatase(ALP), osteocalcin(OC) and bone sialoprotein(BSP) which are markers of osteoblast differentiation. Non-treated group did not show mineralized nodule. CN-C or CN-H-treated group showed minerlaized nodules in 16 days. In northern blot analysis, CN-C or CN-H-treated group showed the elevated expression of ALP, BSP and OC in 16 days. These results suggest the possibility to develop deer antler as a bone regenerative agent in periodontal therapy by showing the stimulating activity of deer antler on differentiation of osteoblast.
Animals ; Antlers* ; Asian Continental Ancestry Group ; Blotting, Northern ; Chloroform ; Deer ; Glucose ; Humans ; Osteoblasts ; Pharmacology ; RNA, Messenger

Scleroderma is a connective tissue disease characterized by excessive accumulation of collagen in skin and visceral organs due to increased collagen production by scleroderma fibroblasts. The basic etiology of this collagen accumulation is not known. We examined the expression of various extracellular matrix genes in cultured fibrolasts using Northern blot and slot-blot hybridization. The scleroderma fibroblasts exhibited characteristic mRNA size of extracellular matrix genes and prominanty increased type I and III procollagen mRNAs levels compared to control fibroblasts cultures from univolved skin. The ratios of type I /IE procollagen in scleroderma cell lines were not so much different to the controls. These results indicate that increases of collagen biosynthesis in scleroderma can be a accounted for, at least in part, by an increased content of transcriptable type I and type JE procollagen mRNAs, both.
Blotting, Northern ; Cell Line ; Collagen ; Connective Tissue Diseases ; Extracellular Matrix ; Fibroblasts* ; Procollagen ; RNA, Messenger ; Skin