Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin.

Calsequestrin (CSQ) is the major Ca2+ binding protein of the cardiac sarcoplasmic reticulum (SR). Transgenic mice overexpressing CSQ at the age of 7 weeks exhibit concentric cardiac hypertrophy, and by 13 weeks the condition progresses to dilated cardiomyopathy. The present study used a differential display analysis to identify genes whose expressions are modulated in the CSQ-overexpressing mouse hearts to provide information on the mechanism of transition from concentric cardiac hypertrophy to failure. Cardiac ankyrin repeat protein (CARP), glutathione peroxidase (Gpx1), and genes which participate in the formation of extracellular matrix including decorin, TSC-36, Magp2, Osf2, and SPARC are upregulated in CSQ mouse hearts at 7 and 13 weeks of age compared to those of non-transgenic littermates. In addition, two novel genes without sequence similarities to any known genes are upregulated in CSQ-overexpressing mouse hearts. Several genes are downregulated at 13 weeks, including SR Ca2+-ATPase (SERCA2) and adenine nucleotide translocase 1 (Ant1) genes. Further, a functionally yet unknown gene (NM_026586) previously identified in the mouse wolffian duct is dramatically downregulated in CSQ mice with dilated hearts. Thus, CARP, Gpx1, and genes encoding extracellular matrix proteins may participate in the development of cardiac hypertrophy and fibrosis, and changes in SERCA2, Ant1, and NM_026586 mRNA expression may be involved in transition from concentric to dilated cardiac hypertrophy.

Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis.

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.

Endothelin-1 induces phosphorylation of GATA-4 transcription factor in the HL-1 atrial-muscle cell line.

The transcription factor GATA-4 plays a central role in the regulation of cardiac-muscle gene transcription. The present study demonstrates that endothelin-1 (ET-1) induces GATA-4 activation and phosphorylation. The treatment of HL-1 adult mouse atrial-muscle cells with ET-1 (30 nM) caused a rapid increase in the DNA binding activity of GATA-4 within 3 min. The activation was associated with an upward mobility shift of the GATA-4 band on native PAGE in an electrophoretic- mobility-shift assay. The upward shift of the GATA-4 band also occurred on SDS/PAGE as monitored by immunoblotting. The in vitro treatment of nuclear extracts with lambda-protein phosphatase abolished the upward shift, indicating that GATA-4 was phosphorylated. ET-1 activated the p44/42 mitogen-activated protein kinase (MAPK) and the MAPK kinase (MEK) within 3 min, and PD98059 (a specific inhibitor of MEK) abolished the ET-1-induced GATA-4 phosphorylation. PMA also caused the rapid activation of MAPK and the phosphorylation of GATA-4. In contrast, the activation of MAPK by phenylephrine or H(2)O(2) was weak and did not lead to GATA-4 phosphorylation. Thus ET-1 induces a GATA-4 phosphorylation by activating a MEK-MAPK pathway.

Effects of thiol antioxidants on hepatocyte growth factor signaling in cardiac myocytes.

We describe here novel antioxidant-sensitive events in which activation kinetics are delayed, leading to inhibition of cell signaling. Hepatocyte growth factor (HGF) transiently phosphorylated p44/42 mitogen-activated protein kinase (MAPK) with a peak at 3-5 min in HL-1 adult cardiac myocytes. Pretreatment of cells with thiol antioxidants, N-acetylcysteine or alpha-lipoic acid attenuated MAPK phosphorylation induced by a 3-min incubation with HGF. However, kinetic analysis revealed that the apparent inhibition of HGF signaling was due to a delay in the activation because HGF phosphorylated MAPK with a peak at 5-7 min in cells treated with thiol antioxidants. This 2-min delay in HGF activation of MAPK resulted in >5-min delay in phosphorylation of MAPK targets such as p90RSK and GATA-4. Hydrogen peroxide did not mimic HGF signaling, and HGF did not induce reactive oxygen species production. Thus, in cardiac myocytes, thiol antioxidants delay HGF-mediated MAPK activation and suppress subsequent signaling eventsvia reactive oxygen species-independent mechanism.

Antitumor protein (AP) from a mushroom induced apoptosis to transformed human keratinocyte by controlling the status of prb, c-MYC, cyclin E-cdk2, and p21WAF1 in the G1/S transition.

Antitumor protein (AP) from a mushroom, induced the morphological changes typical to apoptosis such as nuclear condensation, aneuploidity, and DNA fragmentation at concentrations as low as 5-20 ng/ml to cancer cells. Molecular alterations related to cell cycle. Molecular alterations related to cell cycle, especially G1/S transition were investigated with a human keratinocyte transformed with oncoproteins, E6 and E7 of human pappiloma virus(HPV)-16. AP didn't alter significantly and oncosuppressor p53 level, but induced hyperphosphorylation of pRb. Time-dependent change of G1 cyclins, cdk2 and cdk4 after addition of AP showed that expression level of cdk inhibitors, INK4 family, and p27KIP1 did not altered, while that of p21WAF1 was downregulated.

Regulation of GATA-4 and AP-1 in transgenic mice overexpressing cardiac calsequestrin.

Transgenic mouse hearts overexpressing the Ca(2+)-binding protein calsequestrin (CSQ) have an accompanying 10-fold increase in the sarcoplasmic reticulum (SR) Ca2+ load, however, exhibits slow and small Ca(2+)-induced Ca2+ release. Such slow kinetics of Ca2+ release may have activated excitation-transcription coupling as CSQ overexpressing hearts have induced levels of NFAT and GATA-4 activities and higher levels of c-fos mRNA and cFos protein compared to those of non-transgenic littermates. Adaptive responses, however, appear to downregulate transcriptional regulators controlling c-fos gene including serum response factor and Ca2+/cAMP response element-binding protein. CSQ-overexpressing hearts also had decreased levels of cJun protein, resulting in downregulated AP-1 activity. The mRNA levels of angiotensin II type1a receptor which requires AP-1 and GATA-4 for gene transcription was suppressed in CSQ overexpressing hearts. These results demonstrate that CSQ can regulate GATA-4- and AP-1-dependent transcriptional events, indicating the existence of SR-nuclear circuits of signal transduction in adult cardiac muscle.

Protein Disulfide Isomerase Activity of Some Plant Seeds.

The activity of protein disulfide isomerase, in the extracts of several dormant seeds including soybean, rice, wheat, and maize was assayed. The activity was higher in the extracts of beans than in those of the other seeds. A correlation was significant (R=0.95 and 0.93; p<0.01) between the PDI activity and the concentration of protein soluble in a salt solution.

Effects of prolactins on the chromatophores of the tilapia, Oreochromis niloticus.

Using isolated scales and split-fin preparations of the tilapia Oreochromis niloticus, the effects of a pair of prolactins of the tilapia Oreochromis mossambicus (tPRL177 and tPRL188) and of ovine prolactin (oPRL) on chromatophores were studied in vitro. These peptides caused melanosome aggregation and dispersion of xanthosomes, especially in the split preparations. Their relative effectiveness was as follows: tPRL177 > oPRL > tPRL188. Moreover, tPRL177 at 100 nM induced a high level of pigment dispersion in cultured xanthophores and erythrophores, but tPRL188 at the same concentration did not have this effect. We also examined the responses of chromatophores to oPRL in primary cell culture and found that xanthophores and erythrophores respond to the peptide by pigment dispersion in a dose-dependent manner, whereas cultured melanophores showed little aggregation of pigment. In denervated melanophores in the split-fin preparations, tPRL177 failed to induce aggregation of pigment. From these results, it was concluded that prolactin affects brightly pigmented cells of the tilapia directly, but affects melanophores indirectly. Norepinephrine which might leak from varicosities of chromatic nerve fibers by virtue of the action of prolactin molecules may be responsible for melanosome aggregation.

Comparative studies on the immunosuppressive effect among 5'-deoxy-5-fluorouridine, ftorafur, and 5-fluorouracil.

The immunosuppressive effect of 5'-deoxy-5-fluorouridine (5'-DFUR, Ro 21-9738) was examined for both humoral and cell-mediated immunity in mice in comparison with that of 5-fluorouracil and Ftorafur (FT-207). By both oral and intraperitoneal administration, 5'-DFUR was found to be much less suppressive than 5-fluorouracil and FT-207 in all immune responses tested; hemolysin plaque-forming cells, serum hemolysin titer, delayed-type hypersensitivity and allogeneic tumor rejection. Furthermore, 5'-DFUR did not induce any reduction in either the spleen weight, thymus weight, total spleen cell number, or peripheral leucocyte number.