Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.

In Escherichia coli, all mRNAs are translated by one pool of functionally identical ribosomes.

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Hypoxia-inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their alpha subunits. We have demonstrated that hypoxia stimulates translation of HIF-1alpha and -2alpha proteins by distributing HIF-alpha mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C-alpha (cPKC-alpha), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to approximately 40-50% of HIF-alpha proteins accumulation after 3 h of 1% O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC-alpha inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap-mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium-induced activation of cPKC-alpha hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia-stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.

The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the 3-galactosidase mRNA in Escherichia coli was studied. Of the 39 mutants examined, the level of expression varies over a 20-fold range. The most favorable combinations of bases in the -1 triplet are UAU and CUU. The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU. In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances. In both cases, however, the degree of enhancement depends on its context, i.e. the neighboring bases. Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the 3-galactosidase mRNA.

Abstract-Chronic intermittent hypoxia, a characteristic feature of sleep-disordered breathing, induces hypertension through augmented sympathetic nerve activity and requires the presence of functional carotid body arterial chemoreceptors. In contrast, chronic sustained hypoxia does not alter blood pressure. We therefore analyzed the biosynthetic pathways of catecholamines in peripheral nervous system structures involved in the pathogenesis of intermittent hypoxia-induced hypertension, namely, carotid bodies, superior cervical ganglia, and adrenal glands. Rats were exposed to either intermittent hypoxia (90 seconds of room air alternating with 90 seconds of 10% O 2 ) or to sustained hypoxia (10% O 2 ) for 1 to 30 days. Dopamine, norepinephrine, epinephrine, dihydroxyphenylacetic acid, and 5-hydroxytyptamine contents were measured by high-performance liquid chromatography. Expression of tyrosine hydroxylase and its phosphorylated forms, dopamine ␤-hydroxylase, phenylethanolamine N-methyltransferase, and GTP cyclohydrolase-1 were determined by Western blot analyses. Both sustained and intermittent hypoxia significantly increased dopamine and norepinephrine content in carotid bodies but not in sympathetic ganglia or adrenal glands. In carotid bodies, both types of hypoxia augmented total levels of tyrosine hydroxylase protein and its phosphorylation on serines 19, 31, 40, as well as levels of GTP cyclohydrolase-1. However, the effects of intermittent hypoxia on catecholaminergic pathways were significantly smaller and delayed than those induced by sustained hypoxia. Thus, attenuated induction of catecholaminergic phenotype by intermittent hypoxia in carotid body may play a role in development of hypertension associated with sleep-disordered breathing. The effects of both types of hypoxia on expression of catecholaminergic enzymes in superior cervical neurons and adrenal glands were transient and small. Key Words: blood pressure Ⅲ catecholamines Ⅲ chemoreceptors Ⅲ sympathetic nervous system Ⅲ sleep apnea syndromes Ⅲ oxygen Ⅲ adrenal gland S leep-disordered breathing (SDB) is a frequent condition that affects Ϸ5% of the population and is characterized by repetitive episodes of hypoxemia and hypercapnia, followed by arousal, respiratory recovery, and reoxygenation. Persistent SDB is causally associated with substantial cardiovascular morbidity, the most important being systemic arterial hypertension (for recent comprehensive review, see Fletcher, 2003 1 ). The mechanisms involved in the genesis of this hypertension appear to involve several different components including augmented sympathetic nerve activity, 1-4 altered function of arterial chemoreceptors, 1,5-7 elevated levels of circulating norepinephrine (NE), 8 -10 decreased vascular responses to nitric oxide, 11 increased plasma concentrations of endothelin, 12,13 and altered regulation of the renal kallikrein-kallistatin pathway. 14 The use of animal model systems in which different components of SDB can be studied individually have revealed that intermittent hy...

We have developed a gene expression system in Escherichia coli that contains a portable Shine-Dalgarno region. Transcription of this system is under the direction of a hybrid promoter (tacII) derived from trp and lac-UV5 promoter sequences which is followed by a region that encodes a portable Shine-Dalgarno region (PSDR). Using a series of synthetic PSDRs, we varied the four bases that follow the Shine-Dalgarno (SD) region. We found that the presence of four A residues or four T residues in this position gives the highest translational efficiency. The presence of four C residues reduces the translation efficiency by 50% as compared with PSDRs with A or T residues. The presence of four G residues following the SD region lowers the translational efficiency by at least 75% with respect to PSDRs with A or T residues.

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