Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3P protein as well as for two divergent family members, HFH-1 and HFH-2.We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes.Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3p protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3P with corresponding residues from HFH-1 enabled the HNF-3I recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.Deciphering mechanisms which lead to transcriptional regulation of a distinct array of genes in a particular cell type is critical for understanding cellular commitment during mammalian embryogenesis. Differential expression of protein-encoding genes occurs at the point of transcriptional initiation and involves the assembly of several well-characterized basal factors with TATA-binding protein and RNA polymerase II at the initiation site of the promoter region (17). Promoter and enhancer regions are also composed of multiple DNA sites that interact with sequence-specific transcription factors which are believed to enhance the recruitment of basal factors to the initiation complex. Tissue-restricted gene expression thus relies upon the recognition of multiple cis-acting DNA sequences by cell-specific nuclear factors that potentiate or, in some instances, repress transcriptional initiation (23,28,32). Because transcription factors play a central role in regulating cellular differentiation, the analysis of their molecular structure and expression patterns has been fruitful in elucidating regulatory pathways involved in establishing tissue-specific gene transcription.The functional analysis of a number of transcription factors has demonstrated that they are modular in structure, consisting of independently functioning protein domains (11,(18)(19)(20).

The hepatocyte nuclear factor 3 (HNF-3) gene family is composed of three proteins (a4, 1, and 'y) that are transcription factors involved in the coordinate expression of several liver genes. All three proteins share strong homology in their DNA binding domains (region I) and are able to recognize the same DNA sequence. They also possess two similar stretches of amino acids at the carboxyl terminus (regions H and I) and a fourth segment of homology at the amino terminus (region 1V amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-r domain. Region IV is abundant in serine amino acids and contains two putative casein kinase I phosphorylation sites, a feature similar to protein motifs described for the transcription factors Pit-1/GHF-1 and HNF-1. Cellular differentiation during mammalian development is accompanied by the differential expression of tissue-specific genes (27). The identification of genes responsible for homeotic or developmental mutations in Drosophila melanogaster has shown that temporal cascades of transcription factors are involved in establishment of tissue-specific expression patterns (36,55). The existence of homologies between the homeotic proteins and mammalian transcription factors suggests that similar regulatory cascades are employed during mammalian development (20, 55). Since transcriptional initiation is the main regulatory step in the expression of tissue-specific genes, an understanding of transcriptional control will provide insight into mechanisms of cellular differentiation (27, 45). Tissue-specific gene transcription is maintained through the recognition of promoter sites by transcription factors which display restricted cellular activity (27). The functional characterization of these tissue-specific factors will identify protein regions involved in transcriptional activation and may provide insight into their regulatory mechanisms (3,16,25,35,48,50,57,62). The analysis of promoter regions regulating the expression of tissue-specific transcription factors may identify their position in the hierarchy of the developmental regulatory cascade (2,8,31,41,42,49,58,61,63).The analysis of transcriptional initiation has been the focus of considerable research because it is a major regulatory step in gene expression. The initiation complex is * Corresponding author.composed of an array of basal transcription factors (for example, TFII-A, -B, -D, -E, -F, and -G) that usually assemble at the TATA box sequence with RNA polymerase II (for reviews, see references 27, 45, and 52). In addition to the TATA sequences, promoter regions include multiple DNA binding motifs that bind sequence-specific transcription factors and elevate the rate of transcriptional initiation. The functional analysis of a number of transcription factors has demonstrated that these proteins are modular in structure and consist of domains that can function independently of one another. These analyses have identified r...

In this review, we will examine the most recent preclinical evidence in support of the fact that both acute and chronic stress may have a detrimental impact on the normal function of the dopaminergic system. In recent decades, the term stress has changed its meaning from that of a 'non-specific body response' to a 'monitoring system of internal and external cues'; that is a modality of reaction of the mammalian central nervous system (CNS) which is critical to the adaptation of the organism to its environment. Compelling results have demonstrated that the dopaminergic system is important not only for hedonic impact or reward learning but also, in a broader sense, for reactivity to perturbation in environmental conditions, for selective information processing, and for general emotional responses, which are essential functions in the ability (or failure) to cope with the external world. In this, stress directly influences several basic behaviors which are mediated by the dopaminergic system such as locomotor activity, sexual activity, appetite, and cross sensitization with drugs of abuse. Studies using rat lines which are genetically different in dopamine (DA) physiology, have shown that even small alterations in the birth procedure or early life stress events may contribute to the pathophysiology of psychiatric disorders-in particular those involving central DA dysfunctionand may cause depression or psychotic derangement in the offspring. Finally, the fact that the dopaminergic system after stress responds, preferentially, in the medial prefrontal cortex (MFC), is thought to serve, in humans, as a protection against positive psychotic symptoms, since the increased DA activity in the MFC suppresses limbic DA transmission. However, excessive MFC dopaminergic activity has a negative impact on the cognitive functions of primates, making them unable to select and process significant environmental stimuli. Thus it appears that a critical range of DA turnover is necessary for optimal cognitive functioning after stress, in the response of the CNS to ever-changing environmental demands. Molecular Psychiatry (2000) 5, 14-21.

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The search for new ocular hypotensive agents represents a frontier of current eye research because blindness due to optic neuropathy occurs insidiously in 10% of all patients affected by glaucoma. Cannabinoids have been proposed to lower intraocular pressure by either central or peripheral effects but a specific mechanism for this action has never been elucidated. We recently demonstrated the presence of the central cannabinoid receptor (CB(1)) mRNA and protein in the human ciliary body. In the present study we show that the synthetic CB(1) receptor agonist, WIN 55212--2, applied topically at doses of 25 or 50 microg (n = 8), decreases the intraocular pressure of human glaucoma resistant to conventional therapies within the first 30 min (15 +/- 0.5% and 23 +/- 0.9%, respectively). A maximal reduction of 20 +/- 0.7% and 31 +/- 0.6%, respectively, is reached in the first 60 min. These data confirm that CB(1) receptors have direct involvement in the regulation of human intraocular pressure, and suggest that, among various classes of promising antiglaucoma agents, synthetic CB(1) receptor agonists should deserve further research and clinical development.