Luca Benatti

Molecular cloning of rat kynurenine aminotransferase: Identity with glutamine transaminase K

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of l‐kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS‐1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.

Alternative Splicing at the C-terminal but not at the N-terminal Domain of the NMDA Receptor NR1 is Altered in the Kindled Hippocampus

Several lines of evidence suggest that N‐methyl‐D‐aspartate (NMDA) receptors significantly contribute to the development of kindling. In addition, a lasting enhancement of the NMDA receptor function has been suggested to play a significant role in the chronic hyperexcitability occurring in the hippocampus after kindling epileptogenesis. We have investigated whether hippocampal kindling induces changes in the NMDA receptor at the molecular level by assessing the expression of mRNAs of the different spliced variants at the N‐terminal (exon 5) and C‐terminal (exon 21) position of the NMDA receptor 1 (NR1) gene by means of the reverse transcription‐polymerase chain reaction. Alternative splicing at exon 5 confers different sensitivity of the NMDA receptor to polyamines while exon 21 encodes a 37‐amino acid insert containing the major phosphorylation sites for protein kinase C. One week after the acquisition of stage 5 of kindling in rats (generalized tonic‐clonic seizures), the relative abundance of the two alternatively spliced forms at the C‐terminal domain, respectively containing (+) or lacking (−) exon 21, was reversed compared to controls (implanted with electrodes but not stimulated) in the dorsal hippocampus ipsilateral and contralateral to the electrical stimulation. The exon 21+/exon 21− mRNA ratio for controls was 1.3 ± 0.04 (mean ± SE); for ipsilaterally kindled rats it was 0.64 ± 0.05 (P < 0.05), and for contralaterally kindled rats it was 0.48 ± 0.07 (P < 0.01). Similar bilateral effects were observed in the ventral hippocampus (temporal pole). No changes were found 4 weeks after stage 5 seizures and 1 week after the induction of a single afterdischarge. No significant alterations were induced by kindling in the relative abundance of the spliced variants containing or lacking exon 5. Our findings show selective changes in alternative splicing of the NR1 gene after repeated application of an epileptogenic stimulus. This may generate receptors with different functional properties, which may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

Regulation of Endothelin‐1 Biosynthesisa

The 2 1-amino acid vasoconstrictor peptide endothelin-1 (ET1 ), originally isolated from the culture medium of aortic endothelial cells,’ has been further identified in several other tissue^.^-^ ET-1 has surprisingly potent and wide-ranging biological activities. In particular, given its potential role in the control of vascular tone and the growth of smooth muscle cells, alterations in the regulation of ET-1 biosynthesis may contribute to several aspects of atherosclerotic vascular diseases.6 Therefore, knowledge of the regulation of ET-1 biosynthesis may provide novel pathophysiological insights and suggests potential therapeutic strategies of relevance to vascular diseases. Here we focus on the events controlling preproET-1 mRNA expression and translation, as well as a posttranslational processing and secretion of mature ET-I.

Two preproendothelin 1 mRNAs transcribed by alternative promoters.

Endothelin-1, initially identified as potent vasoconstrictor secreted by vascular endothelial cells, was subsequently found to have many effects on both vascular and nonvascular tissues. We have identified from a human placenta cDNA library a clone (cDNA-2) which corresponds to a novel 5-extended preproendothelin 1 (preproET-1) mRNA. Comparison with the known preproET-1 mRNA (cDNA-1), showed that the two molecules share the same coding sequence but differ in the 5-untranslated region. Interestingly, cDNA-2 extends upstream of promoter regions previously shown to be essential for full preproET-1 expression. Primer extension and PCR analysis of human placenta RNA demonstrated the presence of additional transcription initiation sites located upstream of the previously identified preproET-1 CAP site. Moreover, the two mRNAs show different pattern of expression. To elucidate the mechanisms controlling the production of alternative transcripts we transfected COS-1 cells with a series of preproET-1 promoter deletion mutants. This analysis revealed that the human preproET-1 gene can be transcribed from a proximal and a distal promoter element which has hitherto been undetected. In addition, we demonstrate the presence of a region in the down-epithelial specific expression.

A Saporin-6 cDNA containing a precursor sequence coding for a carboxyl-terminal extension

Saporin‐6 is a single‐chain ribosome inactivating protein (RIP) from the seeds and the leaves of Saponaria officinalis (Caryophyllaceae). Here we have identified the COOH‐terminal end of mature Saporin‐6 and, by cDNA sequencing, the predicted carboxyl‐terminal sequence of a leaf saporin‐6 primary translation product. Our data indicate that the characterized cDNA codes for a precursor contiaing a 22 amino acid carboxyl‐terminal extension, not present in mature Saporin‐6, that shows similarity to carboxyl‐terminal propeptides of vacuolar proteins, suggesting that it may be involved in protein trafficking.

Cloning and recombinant expression of rat and human kynureninase

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse‐transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS‐1 cells were transfected with both cDNAs. The K m values of the rat enzyme, for l‐kynurenine and dl‐3‐hydroxykynurenine, were 440±20 μM and 32±5 μM and of the human enzyme 440±20 μM and 49±6 μM, respectively. Interestingly, COS‐1 cells transfected with the cDNA coding for rat kynureninase also display cysteine‐conjugate β‐lyase activity.

Secretion of biologically active leech hirudin from baculovirus-infected insect cells

The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.

The signal peptide of human preproendothelin-1

Synthetic mRNAs were produced using either the complete coding sequence of a human preproendothelin‐1 cDNA clone or a truncated form in which the portion encoding the first 17 amino acids, representing a putative signal peptide for insertion into the endoplasmic reticulum, was replaced with a methionine codon. The mRNAs were translated in vitro in the presence or in the absence of microsomal membranes. Protection from trypsin digestion demonstrated that the full‐length polypeptide, but not the truncated form, could be inserted into the membranes. Sequence analysis revealed that membrane insertion is accompanied by removal of the first 17 amino acids. These results indicate that the first 17 amino acids of human preproendothelin‐1 are a functional signal peptide which allows the protein to enter the secretory pathway.

ZFM1/SF1 MRNA IN RAT AND GERBIL BRAIN AFTER GLOBAL ISCHAEMIA

Cerebral ischaemia results in significant brain damage, but the molecular mechanisms associated with ischaemia‐induced brain injury are not well defined. We have adopted an improved differential‐display method to search for new ischaemia‐related genes. Among the different cDNAs isolated following transient forebrain ischaemia in rat, PH3.3 was selected for further studies. The search for homologies revealed that it is the rat homologue to human zinc finger motif 1 (ZFM1), also called mammalian splicing factor 1 (SF1). With Northern blot, PH3.3 hybridized with three mRNA species of 2.3, 2.9 and 3.6u2003kb, significantly increased at 6u2003h and 5u2003days after the ischaemic insult. These findings were extended also to another animal model. In situ hybridization in ischaemic gerbils showed that PH3.3 mRNA was induced in the dentate gyrus as early as 4u2003h post‐ischaemia. Expression peaked at 2u2003days in the whole hippocampus and cortex, and then progressively decreased towards sham levels. By day 4, expression had disappeared almost entirely from the cells in the CA1 region of the hippocampus, concomitant with the degeneration of pyramidal neurons. Interestingly, ZFM1/SF1 has been recently identified as activated following p53‐induced apoptosis. Several lines of evidence suggest that p53 may play two roles in the post‐ischaemic brain. The primary role of p53 is to activate DNA repair processes, but if repair fails, apoptosis will be initiated. Thus, ZFM1/SF1 may represent a relevant link between p53 and the neuroprotective/neurodegenerative processes which follow cerebral ischaemia.

Cloning of Rat and Human Kynurenine Aminotransferase

Kynurenic acid (KYNA), a normal component of the mammalian brain, has recently gained attention as a broad spectrum antagonist of ionotropic excitatory amino acid receptors (Stone et al., 1993). While in mammalian peripheral organs, KYNA is biosynthesized from L-kynurenine by several rather unspecific aminotransferases (Noguchi et al., 1975, Okuno et al, 1980), it appears that a single enzyme, kynurenine aminotransferase (KAT), is responsible for KYNA synthesis in the rat brain (Okuno et al., 1991a). In the human brain, on the other hand, two enzymes (KAT I and KAT II) have been characterized (Okuno et al., 1991b). These two KATs have distinct catalytic characteristics and can be physically separated by conventional techniques.