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We determined the nucleotide sequence of a DNA fragment containing the ftsI gene coding for the penicillin-binding protein 3 (PBP-3), an indispensable enzyme for cell division of Escherichia coli. The entire ftsI gene was within the 2.8 kilobase PvuII fragment derived from the chromosomal segment on pLC26-6 (Nishimura et al. 1977). The coding region for PBP-3 was identified by comparison with the N-terminal amino acid sequence of in vitro synthesized PBP-3. The structural gene for ftsI consisted of 1,764 base-pairs coding for a 588 amino acid residue-polypeptide with a molecular weight of 63,850. PBP-3 synthesized in vitro showed a lower mobility in SDS-gel electrophoresis than that of the authentic PBP-3, suggesting that the primary translation product of the ftsI gene may be processed to yield mature PBP-3.

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The developmentally regulated expression of forms of cytochrome P-450, namely, those encoded by lambda HFL33 and NF25 or HLp cDNAs, which were isolated from respective fetal and adult human liver cDNA libraries, was investigated. When EcoRI fragments of cDNA clones of lambda HFL33 and NF25 were used as probes, these probes hybridized with RNA from both fetal and adult human livers. However, when oligonucleotides specific to the coding and 3'-noncoding region of lambda HFL33 (oli-HFL and oli-HFL3', respectively) were used as probes, these probes gave hybridizable bands with RNA from fetal but not adult livers. On the other hand, an oligonucleotide probe specific to the coding region of NF25 and HLp (oli-NF) gave positive bands with RNA only from adult livers. These results indicate that P-450(HFL33) is expressed specifically in fetal livers and that neither P-450NF nor HLp is expressed in fetal livers, but one or both are expressed in adult livers.

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The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.

Abstract-Hypercholesterolemia is a major risk factor involved in abnormal cardiovascular events. Rho-kinase-mediated Ca 2ϩ sensitization of vascular smooth muscle (VSM) plays a critical role in vasospasm and hypertension. We recently identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators for the Rho-kinase-mediated Ca 2ϩ sensitization. Here we report the strong linkage between cholesterol and the Ca 2ϩ sensitization of VSM mediated by a novel SPC/Src-TK/Rho-kinase pathway in both humans and rabbits. The extent of the sensitization correlated well with the total cholesterol or low-density lipoprotein cholesterol levels in serum.However, an inverse correlation with the serum level of high-density lipoprotein cholesterol was observed, and a correlation with other cardiovascular risk factors was nil. When cholesterol-lowering therapy was given to patients and rabbits with hypercholesterolemia, the SPC-induced contractions diminished. Depletion of VSM cholesterol by ␤-cyclodextrin resulted in a loss of membrane caveolin-1, a marker of cholesterol-enriched lipid raft, and inhibited the SPC-induced Ca 2ϩ sensitization and translocation of Rho-kinase from cytosol to the cell membrane. Vasocontractions induced by membrane depolarization and by an adrenergic agonist were cholesterol-independent. Our data support the previously unreported concept that cholesterol potentiates the Ca 2ϩ sensitization of VSM mediated by a SPC/Src-TK/ Rho-kinase pathway, and are also compatible with a role for cholesterol-enriched membrane microdomain, a lipid raft. This process may play an important role in the development of abnormal vascular contractions in patients with hypercholesterolemia. ( Key Words: Ca 2ϩ sensitization Ⅲ contraction Ⅲ membrane lipid raft Ⅲ Rho-kinase Ⅲ sphingosylphosphorylcholine Ⅲ vascular smooth muscle A bnormal vascular contraction as a result of Ca 2ϩ sensitization of vascular smooth muscles (VSMs) has attracted attention as a cause of hypertension and vasospasm. 1 Kureishi et al showed that constitutively active Rho-kinase applied to the cytosol of permeabilized VSM induced Ca 2ϩ sensitization and increased myosin light chain phosphorylation through a mechanism that is independent of a Ca 2ϩ -dependent myosin light chain kinase pathway. 2 In addition, the Rho-kinase blocker Y27632 cures hypertension in rats without affecting normal blood pressure, 3 and it also inhibits vasospasm of coronary arteries in a porcine model. 4 However, the mechanism by which Rho-kinase-mediated Ca 2ϩ sensitization of VSM is triggered in cardiovascular diseases has not been evident.Hyperlipidemia is a major risk factor for abnormal cardiovascular events. Evidence for this is provided by the J-shaped curve showing a strong relationship between serum cholesterol levels and the relative risk for coronary disease 5-7 (see also Figure I in the online data supplement, available at http://circres.ahajournals.org). Atheromatous plaques may be involved in cardiovascular events, 8,9 but the ext...

Background and purpose:The voltage-gated Na + channels (Nav) and their corresponding current (INa) are involved in several cellular processes, crucial to metastasis of cancer cells. We investigated the effects of eicosapentaenoic (EPA), an omega-3 polyunsaturated fatty acid, on INa and metastatic functions (cell proliferation, endocytosis and invasion) in human and rat prostate cancer cell lines (PC-3 and Mat-LyLu cells). Experimental approach: The whole-cell voltage clamp technique and conventional/quantitative real-time reverse transcriptase polymerase chain reaction analysis were used. The presence of Nav proteins was shown by immunohistochemical methods. Alterations in the fatty acid composition of phospholipids after treatment with EPA and metastatic functions were also examined. Key results: A transient inward Na + current (INa), highly sensitive to tetrodotoxin, and NaV proteins were found in these cells. Expression of NaV1.6 and NaV1.7 transcripts (SCN8A and SCN9A) was predominant in PC-3 cells, while NaV1.7 transcript (SCN9A) was the major component in Mat-LyLu cells. Tetrodotoxin or synthetic small interfering RNA targeted for SCN8A and SCN9A inhibited metastatic functions (endocytosis and invasion), but failed to inhibit proliferation in PC-3 cells. Exposure to EPA produced a rapid and concentration-dependent suppression of INa. In cells chronically treated (up to 72h) with EPA, the EPA content of cell lipids increased time-dependently, while arachidonic acid content decreased. Treatment of PC-3 cells with EPA decreased levels of mRNA for SCN9A and SCN8A, cell proliferation, invasion and endocytosis. Conclusion and implications: Treatment with EPA inhibited INa directly and also indirectly, by down-regulation of Nav mRNA expression in prostate cancer cells, thus inhibiting their metastatic potential.