Kindling-induced overexpression of Homer 1A and its functional implications for epileptogenesis.

Despite an extensive research on the molecular basis of epilepsy, the essential players in the epileptogenic process leading to epilepsy are not known. Gene expression analysis is one strategy to enhance our understanding of the genes contributing to the functional neuronal changes underlying epileptogenesis. In the present study, we used the novel MPSS (massively parallel signature sequencing) method for analysis of gene expression in the rat kindling model of temporal lobe epilepsy. Kindling by repeated electrical stimulation of the amygdala resulted in the differential expression of 264 genes in the hippocampus compared to sham controls. The most strongly induced gene was Homer 1A, an immediate early gene involved in the modulation of glutamate receptor function. The overexpression of Homer 1A in the hippocampus of kindled rats was confirmed by RT-PCR. In order to evaluate the functional implications of Homer 1A overexpression for kindling, we used transgenic mice that permanently overexpress Homer 1A. Immunohistochemical characterization of these mice showed a marked Homer 1A overexpression in glutamatergic neurons of the hippocampus. Kindling of Homer 1A overexpressing mice resulted in a retardation of seizure generalization compared to wild-type controls. The data demonstrate that kindling-induced epileptogenesis leads to a striking overexpression of Homer 1A in the hippocampus, which may represent an intrinsic antiepileptogenic and anticonvulsant mechanism in the course of epileptogenesis that counteracts progression of the disease.

The mouse Fkh1/Mf1 gene: cDNA sequence, chromosomal localization and expression in adult tissues.

The 'winged helix' or 'forkhead' transcription factor gene family is defined by a common 100-amino-acid DNA-binding motif. Here, we describe the chromosomal position, start site of transcription, sequence and adult expression pattern of the mouse Fkh1/Mf1 (Forkhead homologue 1/mesoderm/mesenchyme forkhead 1) gene. This gene contains one exon and encodes a protein of 553 amino acids that is highly related to the mouse MFH1 protein. The Fkh1/Mf1 mRNA is expressed widely in adult tissues. Linkage analysis showed that the Fkh1/Mf1 gene is localized to chromosome 13 at 17.02cM from the centromer, in close proximity to Bmp6 and Hfh1, another distinct member of the winged helix gene family.

Targeted disruption of the gene encoding hepatocyte nuclear factor 3gamma results in reduced transcription of hepatocyte-specific genes.

The winged helix transcription factor hepatocyte nuclear factor 3gamma (HNF3gamma) is expressed in embryonic endoderm and its derivatives liver, pancreas, stomach, and intestine, as well as in testis and ovary. We have generated mice carrying an Hnf3g-lacZ fusion which deletes most of the HNF3gamma coding sequence as well as 5.5 kb of 3' flanking region. Mice homozygous for the mutation are fertile, develop normally, and show no morphological defects. The mild phenotype change of the Hnf3g-/- mice can be explained in part by an upregulation of HNF3alpha and HNF3beta in the liver of the mutant animals. Analysis of steady-state mRNA levels as well as transcription rates showed that levels of expression of several HNF3 target genes (phosphoenolpyruvate carboxykinase, transferrin, tyrosine aminotransferase) were reduced by 50 to 70%, indicating that HNF3gamma is an important activator of these genes in vivo.

Expression of the mouse Fkh1/Mf1 and Mfh1 genes in late gestation embryos is restricted to mesoderm derivatives.

We have compared the expression patterns of the mouse Forkhead homologue 1/ mesoderm/mesenchyme forkhead 1 (Fkh1/Mf1) gene with that of the highly related winged helix gene Mfh1 in late gestation mouse embryos. Transcripts for both genes are restricted to derivatives of the mesoderm. Co-expression was found in cartilage primordia of the head, ribs, vertebra and bones. However, in several structures analyzed, Fkh1/Mf1 signals are lower in the inner layers of the developing cartilage than those of Mfh1.

Transcriptional regulation in endoderm development: characterization of an enhancer controlling Hnf3g expression by transgenesis and targeted mutagenesis.

The hepatic nuclear factor 3gamma (Hnf3g) is a member of the winged helix gene family of transcription factors and is thought to be involved in anterior-posterior regionalization of the primitive gut. In this study, cis-regulatory elements essential for the expression of Hnf3g in vivo have been characterized. To this end, a 170 kb yeast artificial chromosome (YAC) carrying the entire Hnf3g locus was isolated and modified with a lacZ reporter gene. The two mouse lines carrying the unfragmented Hnf3g-lacZ YAC showed tissue-specific, copy number-dependent and position-independent expression, proving that 170 kb of the Hnf3g locus contain all elements important in the regulation of Hnf3g. Cis-regulatory elements necessary for expression of Hnf3g were identified in a three-step procedure. First, DNase I hypersensitive site mapping was used to delineate important chromatin regions around the gene required for tissue-specific activation of Hnf3g. Second, plasmid-derived transgenes and gene targeting of the endogenous Hnf3g gene locus were used to demonstrate that the 3'-flanking region of the gene is necessary and sufficient to direct reporter gene expression in liver, pancreas, stomach and small intestine. Third, a binding site for HNF-1alpha and beta, factors expressed in organs derived from the endoderm such as liver, gut and pancreas, was identified in this 3'-enhancer and shown to be crucial for enhancer function in vitro. Based on its expression pattern we inferred that HNF-1beta is a likely candidate for directly activating Hnf3g gene expression during development.

Interaction of mAb to angiotensin-converting enzyme (ACE) with antigen in vitro and in vivo: antibody targeting to the lung induces ACE antigenic modulation.

We previously described that mAb to angiotensin-converting enzyme (ACE), mAb 9B9, accumulates in the rat lungs after systemic injection. In the present work we have documented that mAb 9B9 cross-reacts with human, monkey, rat, cat and hamster ACE, while other ACE antibodies did not cross-react with the rat, cat and hamster enzyme. Anti-ACE mAb 3A5 and I2H5 inhibit human ACE in vitro, while mAb 9B9 does not inhibit ACE activity. Radiolabeled mAb 9B9, but not other antibodies, accumulates selectively in rat, cat and hamster lungs after systemic administration. No accumulation of mAb 9B9 has been observed in hamster kidney, while hamster kidney ACE activity is higher than that in the lung. mAb 9B9 does not induce complement-mediated injury to cultured endothelial cells. No pathological changes were detected in organs of animals after mAb 9B9 injection (10-100 mg/kg). However, injection of these amounts of mAb 9B9 leads to a decrease in ACE activity in the lung homogenates and an increase in serum. In cultured human endothelial cells treatment with mAb 9B9 increases ACE activity in cell medium and decreases in cell lysates. Therefore, while mAb 9B9 does not kill endothelial cells, at high dose it may induce ACE shedding from the cell. The results obtained support the potential of anti-ACE mAb 9B9 for targeting to the lung and for investigations of the pulmonary endothelium.

The HNF-3 gene family of transcription factors in mice: gene structure, cDNA sequence, and mRNA distribution.

The rat HNF-3 (hepatocyte nuclear factor 3) gene family encodes three transcription factors known to be important in the regulation of gene expression in liver and lung. We have cloned and characterized the mouse genes and cDNAs for HNF-3 alpha, beta, and gamma and analyzed their expression patterns in various adult tissues and mouse embryonic stages. The HNF-3 proteins are highly conserved between mouse and rat, with the exception of the amino terminus of HNF-3 gamma, which in mouse is more similar to those of HNF-3 alpha and beta than to the amino termini of the rat HNF-3 gamma protein. The mouse HNF-3 genes are small and contain only two or three (HNF-3 beta) exons with conserved intron-exon boundaries. The proximal promoter of the mouse HNF-3 beta gene is remarkably similar to that of the previously cloned rat HNF-3 beta gene, but is different from the promoters of the HNF-3 alpha and gamma genes. The mRNA distribution of the mouse HNF-3 genes was analyzed by quantitative RNase protection with gene-specific probes. While HNF-3 alpha and beta are restricted mainly to endoderm-derived tissues (lung, liver, stomach, and small intestine), HNF-3 gamma is more extensively expressed, being present additionally in ovary, testis, heart, and adipose tissue, but missing from lung. Transcripts for HNF-3 beta and alpha are detected most abundantly in midgestation embryos (Day 9.5), while HNF-3 gamma expression peaks around Day 15.5 of gestation.

Six members of the mouse forkhead gene family are developmentally regulated.

The 110-aa forkhead domain defines a class of transcription factors that have been shown to be developmentally regulated in Drosophila melanogaster and Xenopus laevis. The forkhead domain is necessary and sufficient for target DNA binding as shown for the rat hepatic nuclear factor 3 (HNF3) gene family. We have cloned six forkhead gene family members from a mouse genomic library in addition to the mouse equivalents of the genes for HNF3 alpha, -beta, and -gamma. The six genes, termed fkh-1 to fkh-6, share a high degree of similarity with the Drosophila forkhead gene, having 57-67% amino acid identity within the forkhead domain. fkh-1 seems to be the mammalian homologue of the Drosophila FD1 gene, as the sequences are 86% identical. fkh-1 to fkh-6 show distinct spatial patterns of expression in adult tissues and are expressed during embryogenesis.

Purification of radiolabeled monoclonal antibodies to angiotensin-converting enzyme significantly improves specificity and efficacy of its targeting into the lung.

The aim of this study was to improve the labeling/purification procedures for monoclonal antibody (MoAb) to angiotensin-converting enzyme (ACE). MoAb 9B9 was very stable upon iodination at a wide range of iodogen concentrations and incubation times, and was also very stable upon storage, indicating the high technological potential of this MoAb. Radiolabeled MoAb 9B9 was purified by (i) adsorption chromatography on cellulose, (ii) HPLC (gel filtration) and (iii) affinity chromatography on ACE-Sepharose. The best result was obtained with cellulose: specificity of MoAb 9B9 accumulation in the lung increased 2-fold. We conclude that the phenomenon of specific lung accumulation of MoAb 9B9 may serve as an ideal (convenient, cheap and technological) assay system for evaluation of monoclonal antibody modification and labeling.

Systemic administration of platelet-activating factor in rat reduces specific pulmonary uptake of circulating monoclonal antibody to angiotensin-converting enzyme.

The biodistribution of radiolabeled mouse monoclonal antibody (MoAb) to angiotensin-converting enzyme (ACE) and control, nonimmune mouse IgG in platelet activating factor (PAF)-treated rats was studied. The blood level of both preparations was slightly decreased (90% of the control) in PAF-treated rats. Specific pulmonary accumulation of anti-ACE MoAb was reduced to 50% of control in contrast to a doubling in nonspecific pulmonary uptake of non-immune IgG. The changes in anti-ACE MoAb biodistribution were lung-specific and were accompanied by decrease in the pulmonary ACE activity (to 60% of control) and increase in serum ACE activity (to 170% of control). Thus anti-ACE MoAb reveals PAF-induced changes in the status of the pulmonary ACE and therefore can be used for the studies of pathology of the pulmonary endothelium.