The maternal diet during pregnancy programs altered expression of the glucocorticoid receptor and type 2 11beta-hydroxysteroid dehydrogenase: potential molecular mechanisms underlying the programming of hypertension in utero.

Potential mechanisms underlying prenatal programming of hypertension in adult life were investigated using a rat model in which maternal protein intake was restricted to 9% vs. 18% casein (control) during pregnancy. Maternal low protein (MLP) offspring exhibit glucocorticoid-dependent raised systolic blood pressure throughout life (20-30 mm Hg above the control). To determine the molecular mechanisms underlying the role of alterations in glucocorticoid hormone action in the prenatal programming of hypertension in MLP offspring, tissues were analyzed for expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11betaHSD1, 11betaHSD2, and corticosteroid-responsive Na/K-adenosine triphosphatase alpha1 and beta1. GR protein (95 kDa) and messenger RNA (mRNA) expression in kidney, liver, lung, and brain was more than 2-fold greater in MLP vs. control offspring during fetal and neonatal life and was more than 3-fold higher during subsequent juvenile and adult life (P < 0.01). This was associated with increased levels of Na/K-adenosine triphosphatase alpha1- and beta1-subunit mRNA expression. Levels of MR gene expression remained unchanged. Exposure to the MLP diet also resulted in markedly reduced levels of 11betaHSD2 expression in the MLP placenta on days 14 and 20 of gestation (P < 0.001), underpinning similar effects on 11betaHSD2 enzyme activity that we reported previously. Levels were also markedly reduced in the kidney and adrenal of MLP offspring during fetal and postnatal life (P < 0.001). This programmed decline in 11betaHSD2 probably contributes to marked increases in glucocorticoid hormone action in these tissues and potentiates both GR- and MR-mediated induction of raised blood pressure. In contrast, levels of 11betaHSD1 mRNA expression in offspring central and peripheral tissues remained unchanged. In conclusion, we have demonstrated that mild protein restriction during pregnancy programs tissue-specific increases in glucocorticoid hormone action that are mediated by persistently elevated expression of GR and decreased expression of 11betaHSD2 during adult life. As glucocorticoids are potent regulators not only of fetal growth but also of blood pressure, our data suggest important potential molecular mechanisms contributing to the prenatal programming of hypertension by maternal undernutrition in the rat.

A bi-functional activator/repressor element required for transcriptional activity of the human UCH-L1 gene assembles a neuron-specific protein: single-strand DNA complex.

The ubiquitin C-terminal hydrolase (UCH)-L1 gene is expressed in a tissue- and cell-specific manner with expression restricted to neurons and neuroendocrine cells. Regulatory DNA sequences from the 5' untranscribed region of the human UCH-L1 gene will promote neuron specific transcription providing that a 59 bp sequence located between nucleotides -182 and -123 is present in reporter gene constructs. We show that this 59 bp sequence is a bi-functional regulator of transcription, acting as an activator in human neuroblastoma cells (SH-SY5Y) and a strong repressor in HeLa cells. The sense strand of the UCH-L1 activator/repressor element can interact with nuclear proteins that recognize single stranded DNA in a sequence specific manner. Nuclear extracts from neuroblastoma cells generate a protein:ssDNA interaction called complex 1B could be converted into a lower mobility complex (1A) by increasing the protein:DNA ratio. This conversion was not observed when using nuclear extracts from HeLa cells. Formation of neuron specific complex 1A could be prevented by incubation of protein:ssDNA complexes at 2 degrees C or in the presence of mM concentrations of MgCl2. In conclusion, we have identified a novel bi-functional regulatory DNA element in the promoter of the human UCH-L1 gene that contributes to neuron-restricted transcription and which can assemble a neuron specific protein:ssDNA complex on its sense strand.

Detection of neuron-specific protein gene product (PGP) 9.5 in the rat and zebrafish using anti-human PGP9.5 antibodies.

Protein gene product (PGP) 9.5 is a developmentally regulated neuron- and neuroendocrine cell-specific ubiquitin carboxy-terminal hydrolase (UCHL1) expressed throughout the mammalian central and peripheral nervous systems. We have compared the use of monoclonal and polyclonal antibodies raised against human PGP9.5 for immunodetection of the protein in tissues of the zebrafish and rat. We show that a monoclonal antibody 13C4, which recognises an N-terminal epitope, detects PGP9.5 on Western blots as a single 27 kDa band present at high levels in zebrafish and rat brain. By contrast, the polyclonal antisera recognises multiple tissue-specific proteins in the rat and fails to detect PGP9.5 in the zebrafish. Finally, we have developed a specific ELISA assay for detection of cellular PGP9.5 using MAb13C4 and have employed the assay to show that PGP9.5 is not upregulated during nerve growth factor (NGF)-induced differentiation of rat PC12 cells.

Amplified expression and large-scale purification of protein L'.

The gene fragment (PPL') encoding the functional unit of peptostreptococcus protein L was isolated using PCR and expressed in E. coli. As the gene fragment lacked its own promoter, the 5' PCR primer was designed to incorporate an Nde1 restriction site (CATATG) into the gene. This enabled the gene to be cloned in frame into an Nde1 restriction site immediately downstream of a trp promoter. To prevent read through, a stop codon was introduced into the 3' primer. Expression of PPL' was up to 27% total cell protein which compares favourably to the 0.1% total soluble cell protein obtained from the original clone of peptostreptococcus. Following a heat step homogeneous PPL' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in yields of 90%.

Identification of evolutionary conserved regulatory sequences in the 5' untranscribed region of the neural-specific ubiquitin C-terminal hydrolase (PGP9.5) gene.

The structure at the 5' end of the gene encoding neural-specific protein gene product 9.5 (PGP9.5) has been compared between two evolutionary distant species: the human and Monodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identity was found across a 1-kb stretch of 5' untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identified a 233-bp CpG-rich minimal promoter. Truncation mutagenesis revealed the presence of essential positive cis-acting regulatory sequences within the region -182 to -123 relative to the transcription initiation site. Sequence alignment analysis of the human and Monodelphis promoter sequences showed 76% identity in this 59-bp region of the gene. A perfectly conserved 12-bp sequence (PSN) located within this region acts as a non-cell-specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detected in human neuroblastoma cell lines but is absent in nonneuronal cell lines such as HeLa. Studies on the cell type specificity of the human PGP9.5 promoter demonstrated that in contrast to the endogenous gene, the promoter is active in HeLa cells. However, the promoter displays higher levels of activity in human neuroblastoma cell lines. A conserved 16-bp sequence located at -356 (motif 5) was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. In conclusion, we have shown that expression of the PGP9.5 gene is regulated by evolutionary conserved positive and negative cis-acting sequences located in the untranscribed region of the gene.

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.

Nucleotide sequence of the gene for peptostreptococcal protein L.

A gene bank of Peptostreptococcus magnus DNA was established using an E. coli host-vector system. Western blot analysis identified a clone expressing protein L which bound to the light chain of human immunoglobulins. DNA sequence determination and analysis revealed an open reading frame of 992 amino acids, giving a theoretical secreted protein of 106 kD with a pl of 4.67.