FR183998, a Na+/H+ exchange inhibitor, suppresses both IL-8 content and myocardial infarct size in a cardiac ischaemia-reperfusion model in rats.

The aim of this study was to determine the effect of FR183998 (5-(2,5-dichlorothiophen-3-yl)-3-[(2-dimethylaminoethyl)carbamoyl]benzoylguanidine dihydrochloride), an Na+/H+ exchange inhibitor, on myocardial interleukin-8 (IL-8) content and myocardial infarct size in a rat ischaemia and reperfusion model. Rats underwent 30 min of ischaemia followed by 1 to 24 h of reperfusion. IL-8 content rapidly increased in reperfused rat hearts. The maximum increase in IL-8 was obtained after 3 h of reperfusion. Intravenous administration of FR183998 at 1 and 3.2 mg kg(-1), 5 min before ischaemia, significantly reduced the IL-8 level after 3 h of reperfusion (122 +/- 16 and 149 +/- 23 pg mg(-1) protein, respectively), compared with that of the saline-treated group (258 +/- 27 pg mg(-1) protein). Myeloperoxidase activity after 3 h of reperfusion was also reduced by FR183998 (from 0.83+0.19 unit g(-1) weight of tissue in the saline-treated group to 0.36 +/- 0.09 and 0.33 +/- 0.06 unit g(-1) weight of tissue in FR183998-treated groups at 1.0 and 3.2 mg kg(-1), respectively). Myocardial infarction induced by 30 min of ischaemia and 24 h of reperfusion was significantly suppressed by the same doses of FR183998 (14.0 +/- 1.5,13.5 +/- 1.9% at 1.0 and 3.2 mg kg(-1)), compared with 22.2+2.7% in the saline-treated group. These results suggestthat IL-8 may contribute to the generation of myocardial infarction in an ischaemia and reperfusion model in rats.

Protective effect of FR183998, a Na+/H+ exchange inhibitor, against postischemic injury after normothermic and prolonged hypothermic ischemia in isolated perfused rat hearts.

Inhibition of Na+/H+ exchanger has been reported to protect hearts from ischemia and reperfusion injury. However, the effect of Na+/H+ exchange inhibition on hypothermic ischemic injury has not been extensively studied and the results are inconsistent. The purpose of this study was to investigate whether inhibition of Na+/H+ exchange with FR183998 (5-(2,5-dichlorothiphen-3-yl)-3-[(2-dimethylaminoethyl)carbamoyl]benzoylguanidine dihydrochloride), a potent Na+/H+ exchange inhibitor, would show protective effects against postischemic cardiac dysfunction after hypothermic as well as normothermic ischemia and furthermore, after hypothermic cardioplegic arrest in isolated rat hearts. FR183998 (3.2 x 10(-8)-3.2 x 10(-7) M) improved post-ischemic recovery of left ventricular developed pressure and suppressed the increase of left ventricular end diastolic pressure in a dose-dependent manner, after not only 45 min of normothermic ischemia but also 6 h of hypothermic ischemia. Furthermore, FRI 83998 (10(-7)-10(-6) M) significantly reduced creatine kinase release during reperfusion after 3 h of hypothermic ischemia with cardioplegia. These results indicate that FR183998 has a potent protective effect on postischemic cardiac dysfunction after normothermic and hypothermic ischemia, and also on reperfusion injury after hypothermic cardioplegic arrest, suggesting that its effect would be additive to cardioplegia.

Amplification and overexpression of TGIF2, a novel homeobox gene of the TALE superclass, in ovarian cancer cell lines.

Homeodomain transcription factors play important roles in directing cellular proliferation and differentiation. A TALE-superclass homeodomain protein, multifunctional repressor of TGFbeta-induced transcription. Here we report identification of TGIF2, a novel TALE-superclass homeodomain protein that shows distinct homology with TGIF, especially in its DNA-binding domain. TGIF2 is expressed ubiquitously in human tissues, with the highest levels being found in heart, kidney, and testis. The TGIF2 product contains a putative nuclear localization signal; translocation of the protein to the nucleus was confirmed by transfection of epitope-tagged cDNA. TGIF2 lies on chromosome 20q11.2-12. Since amplification of 20q is often observed among ovarian cancers, we determined the status of DNA copy-number and expression of TGIF2 in 14 ovarian-cancer cell lines. This gene was over-expressed in all lines that showed amplification by FISH analysis. The results suggested that TGIF2 may play an important role in the development and/or progression of some ovarian tumors through a mechanism of gene amplification.

Genomic organization, sequence and chromosomal localization of the mouse Tbr2 gene and a comparative study with Tbr1.

Members of the T-box family are known to play critical roles in the embryonic development of most animal species. Recently, we have isolated its new mammalian member, Tbr2, from mouse embryonic brain. We have also shown that the expression patterns of Tbr2 and the closely related Tbr1 appear to be reciprocal in the developing brain; Tbr2 is expressed in mesencephalon and rhombencephalon, but expression of Tbr1 is restricted to telencephalon. To investigate possible structural and functional relationships of Tbr2 and other T-box containing genes, we analyzed genomic organization of the murine Tbr2 gene. The Tbr2 gene is composed of six exons (1353, 155, 122, 159, 62 and 1035bp), and five introns (920, 643, 602, 85 and 2036bp). This exon/intron organization is very similar to that of Tbr1. We also analyzed the 3.9kb sequence of the 5' promoter region flanking the Tbr2 gene and the corresponding region of the Tbr1 gene. The sites for Brn-2 and Tst-1 were found in the promoter of Tbr2 but not Tbr1. On the contrary, there were eight HNF-3beta binding sites in the Tbr1 gene promoter but only three in the Tbr2 promoter. The differential presence of putative binding sites for these brain-specific transcription factors may explain the reciprocal expression of Tbr1 and Tbr2. Furthermore, a single chromosomal locus for mouse Tbr2 was assigned to 9F3 by fluorescence in-situ hybridization 1.

Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor.

The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for "kidney-enriched Krüppel-like factor") and the previously isolated MAZ (for "myc-associated zinc finger protein") were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1-luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.

4p- syndrome and 9p tetrasomy mosaicism with cleft lip and palate.

Chromosome 4p- syndrome is a multiple malformation syndrome associated with partial deletion of the short arm of chromosome 4 (4p-). It is characterized by dysmorphic features and retarded development. Cleft lip and/or palate are the major clinical manifestations. Cases of tetrasomy 9p are extremely rare; the principal clinical manifestations of this condition are characteristic craniofacial abnormalities, generalized hypotonia and severe mental retardation. We present the first case of a female infant with 4p deletion and tetrasomy 9p mosaicism, exhibiting a left-sided cleft lip, alveolus and soft palate. Karyotype analysis of lymphocytes cultured from the patient revealed that she was mosaic: 86% of the cells were 46, XX, add (4) (p15.32) and 14% were 47, XX, add (4) (p15.32), +idic (9)(q12). The G-banding pattern appeared consistent with either translocation or partial proximal deletion of 4p. In order to make a definitive cytogenetic diagnosis of isodicentric chromosome 9, fluorescence in situ hybridization (FISH) was applied. At 8 months, when the patient weighed 4.3 kg, her cleft lip was repaired. Before and after surgery there were no seizures, and the postoperative course was uneventful.

Preischemic and postischemic treatment with a new Na+/H+-exchange inhibitor, FR183998, shows cardioprotective effects in rats with cardiac ischemia and reperfusion.

This study describes the pharmacologic profile of a new Na+/H(+)-exchange inhibitor, FR183998, in anesthetized rats. FR183998 had a potent inhibitory effect on Na+/H+ exchange of rat lymphocytes with median inhibitory (IC50) value of 0.3 nM. Treatment with FR183998 (0.01-0.32 mg/kg, i.v.) reduced or completely abolished ventricular fibrillation and mortality induced by 5-min ischemia followed by reperfusion, when it was administered not only 5 min before ischemia but also 1 min before reperfusion. Myocardial infarct size induced by 30-min ischemia and 60-min reperfusion was reduced significantly in a dose-dependent manner by FR183998 (0.1-1.0 mg/kg, i.v.) when the drug was administered preischemically or at an early phase of ischemia. The ventricular tachycardia and the ventricular fibrillation observed during the ischemic period also were suppressed significantly. These results indicate that FR183998 has a strong inhibitory effect on Na+/H+ exchange and suggest that treatment with FR183998 either before or immediately after the onset of ischemia can prevent the occurrence of arrhythmias and myocardial cell necrosis in situations of ischemia and reperfusion.

The human caspase-activated DNase gene (hCAD): genomic structure, exonic single-nucleotide polymorphisms, and a highly polymorphic dinucleotide repeat at the hCAD locus.

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. We determined its genomic structure and identified single-nucleotide polymorphisms (SNPs) within exons 5 and 7, as well as a highly polymorphic dinucleotide repeat of (CT)m(CA)n within the 5' region of the human CAD gene (hCAD). The genomic structure of hCAD presented here, together with information concerning SNPs within the gene, as well as a highly polymorphic (CT)m(CA)n repeat fragment at the hCAD locus, may assist in the construction of genetic maps for exploring gene(s) that play pivotal roles in carcinogenesis.

Protective effect of FR168888, a new Na+/H+ exchange inhibitor, on ischemia and reperfusion-induced arrhythmia and myocardial infarction in rats: in comparison with other cardioprotective compounds.

We have studied the effects of FR168888 (5-hydroxymethyl-3-(pyrrol-1-yl)benzoylguanidine methanesulfonate), a new Na+/H+ exchange inhibitor, on ischemia and reperfusion-induced arrhythmia and myocardial infarction in anesthetized rats and compared them with those of other cardioprotective compounds. FR168888 had a potent inhibitory effect on Na+/H+ exchange of rat lymphocytes acidified with Na+-propionate with a Ki value of 6.4 nM. Pretreatment with FR168888 (0.032-0.32 mg/kg, i.v.) reduced or completely abolished the ventricular fibrillation (VF) induced by reperfusion after 5 min of regional ischemia, while lidocaine, a class I antiarrhythmic agent, showed less effect against VF as compared with FR168888. The size of myocardial infarction induced by 60-min ischemia and 60-min reperfusion was attenuated by FR168888 dose-dependently (1.0-10 mg/kg, i.v.), and ventricular tachycardia and VF were significantly reduced during the ischemic period. In contrast, propranolol and diltiazem did not show such protective effects on myocardial infarct size. In addition, FR168888 did not change hemodynamic parameters in rats. These results indicate that FR168888 has a strong inhibitory effect on Na+/H+ exchange and that treatment with FR168888 can protect the heart from arrhythmia and myocardial cell death in ischemic and reperfused situations.

Human genes for KNSL4 and MAZ are located close to one another on chromosome 16p11.2.

KNSL4 (Kid; kinesin-like DNA-binding protein) is a member of the kinesin family that is involved in spindle formation and the movements of chromosomes during mitosis and meiosis. Myc-associated zinc finger protein (MAZ) participates in both the initiation and the termination of transcription of target genes. We isolated genomic DNA clones that encoded KNSL4 and MAZ from a human cosmid library. Sequence analysis revealed that the two genes were very close to one another. The distance between the two genes was only 1. 2 kb, and this intervening 1.2-kb region was extremely GC-rich. The gene for KNSL4 spanned 16 kb and consisted of 14 exons and 13 introns, while the gene for MAZ spanned 6 kb and consisted of 5 exons and 4 introns. The two genes were mapped to chromosome 16p11.2 by fluorescence in situ hybridization.

Direct activation of endothelial NO pathway by Ba2+ in canine coronary artery.

1. We have reported that Ba2+ causes endothelium-dependent relaxation of canine coronary arteries through NO synthesis in Ca2+-free and depolarizing solution. To determine the cellular mechanisms by which the endothelium-dependent relaxation occurs, we used fura-2 fluorometry (F350 and F390; excitation wavelengths, 350 and 390 nm, respectively) and estimated the intracellular Ba2+ concentration in endothelial and vascular smooth muscle cells. 2. Ba2+ (10(-3) M) increased the fura-2 ratio (F350/F390) recorded from a combined preparation of smooth muscle and endothelium (0.445+/-0.073, n = 4) and contracted the arteries in the presence of 80 mM K+ (0.22+/-0.06 g, n = 4). 3. Diltiazem (3 x l0(-6) M) blocks Ba2+ entry into vascular smooth muscle cells via L-type Ca2+ channels. In this condition, Ba2+ increased the fura-2 ratio in endothelial cells (0.141+/-0.014, n = 5) and relaxed the underlying smooth muscle (0.08+/-0.01 g, n = 5) by a mechanism which was sensitive to 10(-4) M NG-methyl-L-arginine (L-NMMA). 4. Ba2+-induced relaxation was not attenuated with repeated application and was elicited even after endothelium-dependent relaxations in response to 10(-6) M bradykinin were abolished due to tachyphylaxis. Neither 10(-2) M caffeine nor 10(-6) M thapsigargin had effect upon Ba2+-induced relaxation. 5. To further rule out changes in intracellular Ca2+ as a mechanism of Ba2+-induced relaxation, fura-2 fluorescence was measured at the isosbestic wavelengths for Ca2+ (360 nm) and Ba2+ (370 nm) in endothelium-intact arteries. Ba2+ altered F360, but not F370, suggesting little or no contribution of intracellular Ca2+ to the phenomenon of Ba2+-induced relaxation. 6. These results suggest that the Ba2+-induced relaxation is due to its direct activation of endothelial NO synthesis without mobilization of intracellular Ca2+.

Molecular characterization of human Aquaporin-7 gene and its chromosomal mapping.

The cDNA for the seventh mammalian aquaporin (AQP7) was isolated from rat testis, and its expression demonstrated at the tail of late spermatids (Ishibashi et al., J. Biol. Chem. 272 (1997) 20,782-20,786). Here we report the isolation of the mouse and the human AQP7 cDNA and the human AQP7 gene. The human AQP7 gene is identical with human adipose AQP (AQPap or AQP7L). The deduced amino acid sequences of human and mouse AQP7 were 68% and 79% identical to those of rat AQP7, respectively. The mouse AQP7 is 67% identical to the human AQP7. Such a lower conservation of AQP7 among species is unusual in the aquaporin family. The human AQP7 gene is composed of six exons distributing over 6.5 kb. The exon-intron boundaries are identical to those of the human AQP3 gene. The intron sizes are also similar. Moreover, chromosomal localization of AQP7 was assigned to 9p13 by fluorescent in situ hybridization, where AQP3 is also localized, suggesting that 9p13 may be another site of an aquaporin cluster.

cDNA cloning of a short type of multidrug resistance protein homologue, SMRP, from a human lung cancer cell line.

Members of the ATP binding cassette (ABC) superfamily are involved in the energy-dependent transport of a wide variety of substrates including anticancer agents across the membranes. We have cloned a cDNA fragment including a novel ABC sequence from a cisplatin-resistant human lung adenocarcinoma cell line, PC-14/CDDP, by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and screened a cDNA library using the cDNA fragment as a probe. A full-length cDNA clone, BM4.8, was obtained. Sequence analysis showed that the cDNA encoded a short type of multidrug resistance protein homologue, SMRP, by computed homology search. SMRP was composed of 946 amino acids and had two ABCs with walker A and B motifs. This gene was mapped on chromosome 3 at band q27 by fluorescence in situ hybridization (FISH) analysis and was found to be expressed in various tissues by Northern blot analysis.

Assignment of the human gene for KBF2/RBP-Jk to chromosome 9p12-13 and 9q13 by fluorescence in situ hybridization.

The transcription factor KBF2 has been characterized as a factor that binds to the NFkB site of mouse major histocompatibility complex (MHC) class I genes and its amino acid sequence has been shwn to be identical to those of members of the recombination signal-sequence binding protein (RBP-Jk) family. Previous studies by Amakawa et al. (Genomics 17, 306-315, 1993) demonstrated that the functional gene is localized at human chromosome 3q25. However, in the present study we showed by in situ hybridization with the functional KBF2/RBPJk cosmid clone that the gene is localized at 9p12-13 and 9q13, namely, at the same loci as pseudogenes that were reported previously (Zhang et al, Jpn J Human Genet 39, 391-401, 1994).