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

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*

It was previously observed that recombinant flock house virus (FHV) RNA1 was efficiently packaged into turnip yellow mosaic virus (TYMV), provided that the TYMV coat protein (CP) sequence was present at the 3′-end. FHV RNA encapsidated by TYMV CPs also had a four-nucleotide extension at the 5′-end. Since even a short extension at the 5′- and 3′-ends of FHV RNA1 inhibits replication, we examined whether the recombinant FHV RNA is indeed capable of replication. To this end, we introduced constructs expressing recombinant FHV RNAs into the plant Nicotiana benthamiana. Northern blot analysis of inoculated leaves suggested abundant production of recombinant FHV RNA1 and its subgenomic RNA. This demonstrated that recombinant FHV RNA with terminal extensions at both ends was competent for replication. We also showed that the recombinant FHV RNA can express the reporter gene encoding enhanced green fluorescent protein.
Blotting, Northern ; Brassica napus* ; Capsid Proteins* ; Genes, Reporter ; Plants ; RNA* ; Tobacco* ; Tymovirus*

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

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.
Blotting, Northern ; Genome ; Genome Size* ; Plant Viruses ; Product Packaging* ; RNA ; RNA, Viral ; Tobacco ; Tymovirus*

PURPOSE: To investigate the changes of the dopaminergic system in rat striatum after visual deprivation during a critical period of postnatal development. METHODS: The changes of dopamine D1 and D2 receptor (Rc) mRNA were investigated by using Northern blot analysis, in situ hybridization in the rat striatum. The right eyelid of visually deprivated rat was sutured at 10 postnatal days. After visual deprivation for 4 weeks, rats were sacrificed and striatum tissues were removed for analysis. RESULTS: By Northern blot analysis and in situ hybridization, decreased expression of D1 and D2 Rc mRNA was noted in the ipsilateral striatum to the deprivated eye. CONCLUSIONS: These results have shown that visual deprivation during a critical period of postnatal development influences the dopaminergic system in the striatum.
Animals ; Blotting, Northern ; Critical Period (Psychology) ; Dopamine* ; Eyelids ; In Situ Hybridization ; Rats* ; Receptors, Dopamine* ; RNA, Messenger*

PURPOSE: To investigate the change of the Beta adrenergic system in the rat visual cortex and superior colliculus after visual deprivation during a critical period of postnatal development. METHODS: The changes of beta 1 and beta 2 adrenergic receptor mRNA were investigated by using northern blot analysis in the rat visual cortex and superior colliculus. The right eyelid of visually deprived rat was sutured at the 10th postnatal days. After visual deprivation for 4 weeks, the rat were sacrificed and the visual cortex and superior colliculus tissues were removed for analysis. RESULTS: Beta 1 and beta 2 adrenergic receptor mRNA expression was decreased in the contralateral visual cortex to the deprived eye. In the superior colliculus, beta 2 adrenergic receptor mRNA expression increased in both sides, but a much greater increase was shown in the ipsilateral superior colliculus than the contralateral side. CONCLUSIONS: These results suggests that visual deprivation during a critical period of postnatal development influences the beta adrenergic system in the rat visual cortex and superior colliculus.
Animals ; Blotting, Northern ; Critical Period (Psychology) ; Eyelids ; Rats* ; Receptors, Adrenergic ; RNA, Messenger* ; Superior Colliculi* ; Visual Cortex*

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

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*

OBJECTIVETo investigate the expression of thymosin beta10 (Tbeta10) and related changes of actin filament organization in human tumor cell lines with different metastatic potential.

METHODSFour groups of nine human tumor cell lines with different metastatic potential were analyzed for the expression of Tbeta10 mRNA detected by northern-blot and its peptide by immunohistochemical staining. The filamentous actin (F-actin) was stained with TRITC-phalloidin to detect changes in actin organization.

RESULTSIn comparison with the non and/or weakly metastatic counterparts, Tbeta10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. TRITC-phalloidin staining revealed less actin bundles and a fuzzy network of shorter filaments in the highly metastatic tumor cells, while in the non and/or weakly metastatic cancer cell lines, there were thick and orderly arranged actin filaments.

CONCLUSIONSTbeta10 levels correlate positively with the metastatic phenotype in human tumors currently examined. The increased metastatic potential of tumor cells is accompanied by the loss of F-actin and poorly organized actin skeleton. There is a consistent correlation between the elevated Tbeta10 expression and the disrupted actin skeleton.