David Pribnow

Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.

Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.

Determinants contributing to estrogen-regulated expression of SK3

The rSK3 gene encodes a small conductance Ca(2+)-activated K(+) channel that is transcriptionally regulated by estrogen. To examine determinants of rSK3 gene expression, the CAP site was defined and the promoter was identified. A 33 bp sequence adjacent to the promoter was shown to act as an enhancer in L6 cells that express SK3 and estrogen receptor alpha (ER alpha). The 33 bp enhancer was unable to stimulate transcription in Cos7 cells that do not express SK3 or ER alpha. However when cotransfected with ER alpha and stimulated with 17 beta-estradiol (E2) the enhancer was activated. Interestingly, expression of ER alpha in Cos7 cells and E2 treatment was sufficient to induce expression of the endogenous SK3 gene. Only Sp1 and Sp3 specifically bound to the enhancer from Cos7 and L6 nuclear extracts, however, ER alpha increased Sp1s affinity for the enhancer. The data suggest that estrogen regulates SK3 gene expression through interactions between ER alpha and Sp1.

Lignin peroxidase from the basidiomycete Phanerochaete chrysosporium is synthesized as a preproenzyme

The cDNA clone L18 encoding lignin peroxidase LiP2, the most highly expressed LiP isozyme from Phanerochaete chrysosporium strain OGC101, was isolated and sequenced. Comparison of the cDNA sequence with the N-terminal sequence of the mature LiP2 protein isolated from culture medium suggests that the mature protein contains 343 amino acids (aa) and is preceded by a 28-aa leader sequence. In vitro transcription followed by in vitro translation and processing by signal peptidase resulted in cleavage at a site following the Ala21 (counted from the N-terminal Met1 of the initial translation product). The resultant protein contains a 7-aa propeptide, indicating that LiP is synthesized as a preproenzyme.

Skeletal muscle and small‐conductance calcium‐activated potassium channels

Skeletal muscle becomes hyperexcitable following denervation and when cultured in the absence of nerve cells. In these circumstances, the bee venom peptide toxin apamin, a blocker of small‐conductance calcium‐activated potassium (SK) channels, dramatically reduces the hyperexcitability. In this report, we show that SK3 channels are expressed in denervated skeletal muscle and in L6 cells. Action potentials evoked from normal innervated rat skeletal muscle did not exhibit an afterhyperpolarization, indicating a lack of SK channel activity; very low levels of apamin binding sites, SK3 protein, or SK3 mRNA were present. However, denervation resulted in apamin‐sensitive afterhyperpolarizations and increased apamin binding sites, SK3 protein, and SK3 mRNA. Cultured rat L6 myoblasts and differentiated L6 myotubes contained similar levels of SK3 mRNA, although apamin‐sensitive SK currents and apamin binding sites were detected only following myotube differentiation. Therefore, different molecular mechanisms govern SK3 expression levels in denervated muscle compared with muscle cells differentiated in culture.

Angiotensin type 1 receptor antagonism with irbesartan inhibits ventricular hypertrophy and improves diastolic function in the remodeling post-myocardial infarction ventricle.

To evaluate the role of angiotensin II (AII) on diastolic function during post-myocardial infarction (MI) ventricular remodeling, coronary ligation or sham operation was performed in male Sprague-Dawley rats. Experimental animals were maintained on either irbesartan, a selective AT1-receptor antagonist, or no treatment. Measurement of cardiac hypertrophy, diastolic function, and sarcoendoplasmic reticulum adenosine triphosphatase (ATPase; SERCA) and phospholamban (PLB) gene expression was assessed at 6 weeks after MI. Myocardial infarction caused a significant increase in myocardial mass and left ventricular (LV) filling pressure, whereas LV systolic pressure and +dP/dt were reduced. The time constant of isovolumic relaxation (tau) was markedly prolonged after MI. Post-MI hypertrophy was associated with substantial increases in the messenger RNA (mRNA) expression of atrial natriuretic peptide (ANP), but no significant changes in SERCA or PLB levels. Although irbesartan treatment did not significantly alter post-MI LV systolic or filling pressures, it nevertheless effectively decreased ventricular hypertrophy, improved tau, and normalized ANP expression. These results demonstrate that AT1-receptor antagonism has important effects on myocardial hypertrophy and ANP gene expression, which are independent of ventricular loading conditions. In addition, the improvement in diastolic function was not related to changes in SERCA and PLB gene expression, suggesting that enhanced myocardial relaxation was related to the blockade of AII effects on myocyte function or through a reduction of ventricular hypertrophy itself or both.

Complex interactions with direct repeats of a mitogen-responsive VL30 enhancer.

The RVL-3 VL30 enhancer is an LTR-derived triple direct repeat of 35 base pairs that mediates gene induction in response to several different intracellular signaling pathways. Using mobility shift assays, methylation interference and DNase I footprinting, we have investigated the physical interactions between the RVL-3 enhancer and components of nuclear extracts from Rat-1 cells. Each enhancer repeat unit contains a single binding site. Our studies suggest that binding to the double or triple repeat enhancer is cooperative, involving simultaneous occupation of two sites, with a preference for adjacent sites. Binding cooperativity would have implications for the mechanism of gene activation directed from the native VL30 enhancer.