Very closely related short sequences are present at the 5′ end of cytoplasmic mRNAs in Euglena as evidenced by comparison of cDNA sequences and hybrid‐arrested translation experiments. By cloning Euglena gracilis nuclear DNA and isolating the rbcS gene (encoding the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase), we have shown that the short leader sequence does not flank the nuclear gene sequence. The leader sequences were found to constitute the 5′ extremities of a family of small RNAs. Sequencing six members of this family revealed a striking similarity to vertebrate U snRNAs. We propose that a trans‐splicing mechanism transfers the spliced leader (SL) sequence from these small RNAs (SL RNAs) to pre‐mature mRNAs. Transfer of leader sequences to mRNAs by trans‐splicing has been shown only in trypanosomes where cis‐splicing is unknown, and in nematodes where not more than 10% of the mRNAs have leader sequences. Our results strongly suggest that Euglena is a unique organism in which both a widespread trans‐splicing and a cis‐splicing mechanism co‐exist.

Stimulation of beta(2)-AR is necessary for nortriptyline to exert its antiallodynic action against neuropathic pain. These findings provide new insight into the mechanism by which antidepressants alleviate neuropathic pain. Our results also raise the question of a potential incompatibility between beta-blockers that affect beta(2)-AR and antidepressant drugs in patients treated for neuropathic pain.

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A cDNA clone containing the complete human a1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the a1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage A (PL) and initiation of translation at the A cli gene ribosome-binding site. This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein. The polypeptide produced was recognized by antisera raised against human a1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.a1-Antitrypsin is a serum antiprotease of hepatic origin whose most important physiological role is to restrict neutrophil elastase activity in the lung (1-2). A deficiency of a1-antitrypsin upsets the alveolar protease-antiprotease balance, leading to elastase-mediated tissue destruction and chronic pulmonary emphysema (3). Inherited a1-antitrypsin deficiency occurs at a high frequency in European populations (1 in 750 for the two principal variants Z and S) (4). The most common clinically significant variant (type Z) has a single amino acid substitution that is associated with reduced glycosylation of the a1-antitrypsin molecule (5-6). This results in its accumulation in hepatocytes and a reduction in serum concentration to 10-15% of normal (7). Cigarette smoking, a major factor in nonhereditary emphysema, also causes a protease-antiprotease imbalance in the lower respiratory tract (8) as a consequence of both increased elastase levels and a 50% reduction in active alveolar a1-antitrypsin (9)(10). Clinical trials have shown that a1-antitrypsin deficiency can be treated by replacement therapy (11), but the problems of possible viral contamination associated with the use of human blood products deter extensive clinical use of a1-antitrypsin purified from serum. To circumvent this problem we have used the techniques of genetic engineering to produce human a1-antitrypsin in a microorganism. The availability of information on the sequences of baboon and human a1-antitrypsin cDNA clones (12, 13) and the structures of normal and variant genes (14, 15) enabled us to isolate a full-length human a1-antitrypsin cDNA clone. Transfer of this sequence into a high-level expression system resulted in a recombinant E. coli strain capable of synthesizing a1-antitrypsin at levels of up to 15% of total cell protein. MATERIALS AND METHODSBacterial Strains and Plasmids. cDNA banks were prepared using E. coli strain 1106 (supE hsdS met supF). Strain TGE900 [F-su-ilv-bio (XcI857ABamAHI)], which produces the temperature-sensitive XcI857 repressor, was used as host for the PL-containing plasmids.pTG603 is a pBR322 derivative containing a human a1-antitrypsin cDNA insert. pTG920 is the PL-containing expression vector and pTG922 is a derivative that expresses human a1-antitrypsin.Isolation of a1-...

The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components. The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal. Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking. Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein. To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA. We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli. The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation. The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor. The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder.

In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration.