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 3.6 kb, significantly increased at 6 h and 5 days 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 4 h post-ischaemia. Expression peaked at 2 days 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.

Kynurenine aminotransferase I (KATI) isoform gene expression in the rat brain: an in situ hybridization study.

Kynurenine aminotransferase I (KATI) converts kynurenine into kynurenic acid (KYNA), a broadspectrum antagonist at ionotropic excitatory amino acid receptors. The main interest in KYNA centers on its potential neuroprotective action in physiological and pathological conditions. We show here by in situ hybridization that KATI mRNA is widely expressed throughout the adult rat brain. A strong autoradiographic signal was detected in the hippocampus, piriform cortex, and choroid plexus. Microscopic evaluation suggested that KATI mRNA was expressed not only in astrocytes but also in hippocampal neurons and in choroid plexus epithelial cells. Neuronal expression of KATI mRNA was further confirmed by RT-PCR and in a model of transient cerebral ischemia. The expression pattern of the mitochondrial form (mKATI) of the enzyme was almost comparable to that of KATI. The major difference was observed in the choroid plexus where mKATI mRNA signal was very low.

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.

Growth abnormalities in cultured mesangial cells from rats with spontaneous glomerulosclerosis.

Age-related glomerulosclerosis (GS) occurs in normotensive rats of the Milan strain (MNS), but not in genetically-matched hypertensive animals (MHS). Altered mesangial cell (MC) proliferation and matrix expansion are common features of the glomerular scarring process. We evaluated proliferation and matrix protein synthesis of cultured MC from MNS and MHS animals aged 1 and 8 months, that is, before and after the occurrence of GS. [3H]-thymidine (TdR) incorporation into DNA of MC from MNS rats stimulated by 10% FBS serum increased with donor aging from 115 +/- 6.0 to 176 +/- 15, P < 0.01 (% cpm/well over quiescent controls +/- SEM). Under the same experimental conditions, cell counts changed from 101 +/- 4.0 to 146 +/- 5.0, P < 0.01 (% cells/well over quiescent controls). Additionally, cytosolic Ca2+ concentration ([Ca2+]i) rised from 115 +/- 19 to 220 +/- 32 nM and from 112 +/- 24 to 734 +/- 136 nM when fura-2-loaded cells from young and old MNS rats, respectively, were stimulated with 1% FBS. The rate of collagen production also increased with donor age, as well as collagen IV and laminin B1 mRNA expression. In contrast, in MC from MHS rats both DNA synthesis and cell replication rate declined as function of donor age. No differences in the [Ca2+]i responses to FBS were observed, nor collagen production changed with MHS rat senescence. We conclude that the age-associated decline of proliferative activity in MC from MHS animals could actually reflect a normal process of cell aging, possibly protecting from the occurrence of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Agents combining thromboxane receptor antagonism with thromboxane synthase inhibition: [[[2-(1H-imidazol-1-yl)ethylidene]amino]oxy]alkanoic acids.

A new class of compounds combining thromboxane-A2 (TxA2) receptor antagonism and thromboxane synthase inhibition is described. A first series of (E)- and (Z)-[[[2-(1H-imidazol-1-yl)ethylidene]amino]oxy]pentanoic acids showed relevant thromboxane synthase inhibition associated with weak TxA2 receptor antagonism, while a series of (+/-)-(E)-[[[2-(1H-imidazol-1-yl)-3-phenylpropylidene]amino]oxy] pentanoic acids, structurally derived from the former, showed potent and well-balanced dual activity. Structural requirements for significant single and dual activity are discussed. Two close congeners of the latter series, (+/-)-(E)-5-[[[1-cyclohexyl-2-(1H-imidazol-1-yl)-3- phenylpropylidene]amino]oxy]pentanoic acid 23c and its p-fluorophenyl analog 23m, inhibited TxB2 production in vitro, in rat whole blood during clotting, with IC50 of 0.06 and 0.37 microM and antagonized the binding of [3H]SQ 29548 to washed human platelets, with IC50 of 0.08 and 0.02 microM, respectively. These two compounds were selected for further pharmacological evaluation and were shown to antagonize U46619-induced platelet aggregation in human platelet rich plasma with IC50 of 0.30 and 0.44 microM, respectively. They were both orally available, and in particular 23m caused a long lasting ex vivo TxA2 synthase inhibition in the fed rat. The levorotatory enantiomer of 23c, stereospecifically synthesized as a model compound, was found to be more potent than racemic 23c with regard to TxA2 receptor antagonism (IC50 = 0.04 microM) and equivalent to the latter with regard to TxA2 synthase inhibition. A molecular modeling study concerning the levorotatory enantiomer of 23c (S), TxA2, and representative TxA2 antagonists of different classes led to the definition of a putative pharmacophoric model for the TxA2 receptor ligands.

In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.

Human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells.

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.

Heteroduplex morbidity assay and physiolgenetic analysis for the V3 region of HIV-1 isolates from Gulu-Northern Uganda

"In order to identify HIV-1 strains prevalent in Northern Uganda; peripheral blood mononuclear cells (PBMC) of 19 asympotomatic seropositive pregnant women from the District of Gulu-Northern Uganda have been analysed. A 700bp fragment of the HIV-1 env gene; including the V3-V5 region; amplified by Polymerase chain Reaction (PCR) from 10 samples (52.6); was subjected to both heteroduplex Mobility Assay (HMA); for genetic subtyping; and DNA sequence analysis; for nucleotide comparison and phylogenetic studies. The results show the presence of HIV-1 ""A"" and ""D"" subtypes/clades with a strong prevalence; in this rural area; of the ""A""(8/10) over the ""D"" subtype (2/10) unlike what was previously reported in Uganda. By pairwise comparison analysis; the percentage of sequence divergence is low among samples within each subtype (average inter-subtypes divergence of 23). At the aminoacidic level; the two HIV-1 groups are clearly distinct by a tetrameric GPGQ sequence at the V3 loop apex for the A and a GPGR sequence for the D clade. In addition; 10 out of the 19 viral samples (52.6) have been isolated in vitro and 9 of them have been classified as Rapid/High (R/H); showing the high in vitro replication capacity to field isolated also when obtained from asymptomatic individuals. These data; even though on a limited sample size; suggest that in Uganda HIV-1 isolates can be prevalently grouped in 2 major clades; and the comparison of Gulu HIV-1 sequences with the two consensus sequences (Group A and B) identified by Albert et al 1990; indicates that in a 4-year period no major genetic shifts have occurred. These results should be extremely relevant for Uganda future HIV-1 vaccine programs. Supported by Ministero Italiano Sanita (Ric.Corr.1994) and ISDC-World Laboratory; Lausanne (Project MCD-2/7)."