Sally J. Dawson

The status of PAI-1 as a risk factor for arterial and thrombotic disease: A review

Plasminogen activator inhibitor-1 (PAI-1) is a rapid inhibitor of tissue plasminogen (tPA) in vivo. Evidence suggests that the level of plasma PAI-1 activity is responsible for the regulation of the whole fibrinolytic process through this tPA/PAI-1 interaction. Levels of PAI-1 have therefore emerged as a candidate for a thrombotic risk factor. Recent epidemiological data supports the view that high plasma levels of PAI-1 may be important in the pathogenesis of arterial and thrombotic disease. These data are reviewed and their significance discussed. PAI-1 expression has been shown to be regulated by many different factors in vitro and the relevance of these data to in vivo physiology is addressed. The current knowledge of the biochemistry, expression and genetics of PAI-1 is also presented and the significance of this to disease pathogenesis is discussed.

The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins.

The Brn-3a POU family transcription factor is expressed only in posmitotic neurons in the central nervous system and identifies the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development. This factor is also induced when undifferentiated proliferating ND7 cells cease dividing and differentiate to a mature neuronal-like phenotype bearing numerous neurite processes. We show that overexpression of Brn-3a in undifferentiated ND7 cells induces a mature neuronal phenotype characterized by process outgrowth and the induction of genes encoding synaptic proteins, although the cells continue to proliferate. In contrast, the closely related factors Brn-3b and Brn-3c do not have this effect. Although the N-terminal activation domain of Brn-3a is required for maximum induction of neurite outgrowth and gene expression, these effects are primarily dependent on the DNA binding POU domain, which also acts as an activation domain. Overexpression of the isolated POU domain of Brn-3a is sufficient to induce neurite outgrowth, while the ability of full-length Brn-3a to do so is abolished by mutating a single amino acid in the Brn-3a POU homeodomain to its equivalent in Brn-3b. Thus, Brn-3a appears to play a critical role in the specification of the mature neuronal phenotype, acting by stimulating the expression of genes whose products are required for process outgrowth and synapse formation.

Repression of a herpes simplex virus immediate-early promoter by the Oct-2 transcription factor is dependent on an inhibitory region at the N terminus of the protein.

The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.

A Single Amino Acid Change Converts an Inhibitory Transcription Factor into an Activator

The closely related POU family transcription factors Brn-3a and Brn-3b differ in their functional activity with Brn-3a activating several target promoters, which are repressed by Brn-3b. Brn-3b also prevents promoter activation by Brn-3a. Here we have altered a single isoleucine residue in the POU homeodomain of Brn-3b to the valine residue found at the equivalent position in Brn-3a. This change not only abolishes the ability of Brn-3b to repress basal and Brn-3a-stimulated promoter activity but also converts it to an activator of similar potency to Brn-3a. Hence a single amino acid difference determines the difference between an activator and a repressor in the Brn-3 family.

Differential Regulation of the Two Neuronal Nitric-oxide Synthase Gene Promoters by the Oct-2 Transcription Factor

The Oct-2 transcription factor has been shown previously to repress both the cellular tyrosine hydroxylase and the herpes simplex virus immediate-early genes in neuronal cells. Here we identify the gene encoding the neuronal nitric-oxide synthase (nNOS) as the first example of a gene activated in neuronal cells by Oct-2. The levels of the nNOS mRNA and protein are greatly reduced in neuronal cell lines in which Oct-2 levels have been reduced by an antisense method, although these cells have enhanced levels of tyrosine hydroxylase. Moreover, the nNOS gene regulatory region is activated by Oct-2 expression vectors upon cotransfection into both neuronal and non-neuronal cells, and this response is dependent upon a 20-amino acid region within the COOH-terminal activation domain of Oct-2. Of the two closely linked promoters that drive nNOS gene expression, only the downstream 5.1 promoter is activated by Oct-2, whereas the 5.2 promoter is unaffected. These effects are discussed in terms of the potential role of Oct-2 in regulating nNOS expression in the nervous system.

Differential regulation of genes encoding synaptic proteins by members of the Brn-3 subfamily of POU transcription factors.

The three members of the Brn-3 subfamily of POU transcription factors have distinct effects on target gene expression. We show that the promoter of the gene encoding the presynaptic nerve terminal protein SNAP-25 resembles previously characterised target genes in being activated by Brn-3a and Brn-3c, but being repressed by Brn-3b. Unlike other target genes, however, the SNAP-25 promoter can be activated by either the N- or C-terminal activation domains of Brn-3a. In contrast to the SNAP-25 gene, the gene encoding the synaptic vesicle protein synapsin 1 is activated by all the Brn-3 factors, the first gene for which this activation pattern has been reported Interestingly, however, similar activation by all three Brn-3 factors can be observed if the SNAP-25 promoter is truncated by removal of sequences from -2200 to -288 relative to the transcriptional start site. Moreover, a region of the SNAP-25 promoter from -283 to -126 can render a heterologous promoter responsive to activation by all three Brn-3 factors. Differences in promoter structure may thus result in differences in the response to different Brn-3 factors, thus allowing these factors to produce diverse activation patterns of neuronally expressed genes, such as those encoding different synaptic proteins.

High-frequency audiometry reveals high prevalence of aminoglycoside ototoxicity in children with cystic fibrosis

BACKGROUND Intravenous aminoglycoside (IV AG) antibiotics, widely used in patients with cystic fibrosis (CF), are known to have ototoxic complications. Despite this, audiological monitoring is not commonly performed and if performed, uses only standard pure-tone audiometry (PTA). The aim of this study was to investigate ototoxicity in CF children, to determine the most appropriate audiological tests and to identify possible risk factors. METHODS Auditory assessment was performed in CF children using standard pure tone audiometry (PTA), extended high-frequency (EHF) audiometry and distortion-product otoacoustic emissions (DPOAE). RESULTS 70 CF children, mean (SD) age 10.7 (3.5) years, were recruited. Of the 63 children who received IV AG, 15 (24%) children had ototoxicity detected by EHF audiometry and DPOAE. Standard PTA only detected ototoxicity in 13 children. Eleven of these children had received at least 10 courses of IV AG courses. A 25 to 85 dBHL hearing loss (mean±SD: 57.5±25.7 dBHL) across all EHF frequencies and a significant drop in DPOAE amplitudes at frequencies 4 to 8 kHz were detected. However, standard PTA detected a significant hearing loss (>20 dBHL) only at 8 kHz in 5 of these 15 children and none in 2 subjects who had significantly elevated EHF thresholds. The number of courses of IV AG received, age and lower lung function were shown to be risk factors for ototoxicity. CONCLUSIONS CF children who had received at least 10 courses of IV AG had a higher risk of ototoxicity. EHF audiometry identified 2 more children with ototoxicity than standard PTA and depending on facilities available, should be the test of choice for detecting ototoxicity in children with CF receiving IV AG.

Inhibition of neuronal process outgrowth and neuronal specific gene activation by the Brn-3b transcription factor

The differentiation of the ND7 neuronal cell line to a nondividing phenotype bearing numerous neurite processes is accompanied by a dramatic increase in the levels of the activating POU family transcription factor Brn-3a and a corresponding fall in the levels of the closely related inhibitory factor Brn-3b. We have previously shown that the artificial overexpression of Brn-3a in these cells can induce neurite outgrowth and the activation of genes encoding synaptic vesicle proteins in the absence of a differentiation-inducing stimulus. Here we show that overexpression of Brn-3b can reduce process outgrowth and synaptic vesicle gene expression following exposure to a stimulus which would normally induce differentiation. These inhibitory effects are abolished by altering a single amino acid in the POU homeodomain of Brn-3b to its equivalent in Brn-3a. The converse mutation in Brn-3a allows it to inhibit process outgrowth in response to a differentiation-inducing stimulus. Hence a single amino acid difference results in these closely related factors having opposite effects and allows the balance between them to regulate differentiation.

Estrogen-related receptor gamma and hearing function: evidence of a role in humans and mice

Since estrogen is thought to protect pre-menopausal women from age-related hearing loss, we investigated whether variation in estrogen-signalling genes is linked to hearing status in the 1958 British Birth Cohort. This analysis implicated the estrogen-related receptor gamma (ESRRG) gene in determining adult hearing function and was investigated further in a total of 6134 individuals in 3 independent cohorts: (i) the 1958 British Birth Cohort; (ii) a London ARHL case-control cohort; and (iii) a cohort from isolated populations of Italy and Silk Road countries. Evidence of an association between the minor allele of single nucleotide polymorphism (SNP) rs2818964 and hearing status was found in females, but not in males in 2 of these cohorts: p = 0.0058 (London ARHL) and p = 0.0065 (Carlantino, Italy). Furthermore, assessment of hearing in Esrrg knock-out mice revealed a mild 25-dB hearing loss at 5 weeks of age. At 12 weeks, average hearing thresholds in female mice(-/-) were 15 dB worse than in males(-/-). Together these data indicate ESRRG plays a role in maintenance of hearing in both humans and mice.

Aminoglycoside antibiotics cochleotoxicity in paediatric cystic fibrosis (CF) patients: A study using extended high-frequency audiometry and distortion product otoacoustic emissions

Abstract Despite known ototoxic effects of aminoglycoside (AG) antibiotics, audiological assessment is not routinely undertaken in UK CF patients. Consequently, the incidence of hearing loss is not well established. Objective: To document the incidence of hearing loss in cystic fibrosis (CF) children. Design: Hearing function of 45 children from Great Ormond Street Hospital was assessed using pure-tone audiometry up to 20kHz and DPOAEs up to 8kHz. Study Sample: 39/45 of participants had received intravenous (IV) AGs, 23 of which received repeated IV AGs every 3 months. Results: In this high exposure group, 8 (21%) had clear signs of ototoxicity; average 8-20kHz thresholds were elevated by ∼50dB and DPOAE amplitudes were >10dB lower at f2 3.2-6.3 kHz. The remaining 31/39 (79%) of AG exposed patients had normal, even exceptionally good hearing. The 21% incidence of ototoxicity we observed is substantial and higher than previously reported. However, our finding of normal hearing in children with equal AG exposure strongly suggests that other unknown factors, possibly genetic susceptibility, influence this outcome. Conclusions: We recommend comparable auditory testing in all CF patients with high AG exposures. Genetic analysis may help explain the dichotomy in response to AGs found. Sumario A pesar de los conocidos efectos ototóxicos de los antibióticos aminoglucósidos (AG), en el Reino Unido no se lleva a cabo de rutina una evaluación audiológica en los pacientes con fibrosis quística (CF); consecuentemente, la incidencia de hipoacusia no está bien establecida. Objetivo: Documentar la incidencia de trastornos auditivos en niños con fibrosis quística (CF). Diseño: Se evaluó la función auditiva de 45 niños del Hospital Great Ormon Street, usando audiometría de tonos puros hasta 20kHz y DPOAE hasta 8 kHz. Muestra Del Estudio: 39/45 participantes habían recibido AG intravenosos (IV), 23 de los cuáles los recibieron repetidamente por vía IV, cada 3 meses. Resultados: En este grupo de alta exposición, 8 niños (21%) mostraban claros signos de ototoxicidad; los umbrales promedio de 8-20 kHz estaban elevados por ∼50dB y las amplitudes de las DPOAE estaban >10dB más bajas en f2 3.2-6.3 kHz. Los restantes 31/39 (79%) pacientes expuestos a AG tenían una audición normal e incluso, excepcionalmente buena. La incidencia de ototoxicidad del 21% que observamos es sustancial y es más alta de lo previamente reportado. Sin embargo, nuestro hallazgo de audición normal en niños con exposición equivalente a AG sugiere fuertemente que otros factores desconocidos, posiblemente la susceptibilidad genética, influyen en los resultados. Conclusiones: Recomendamos evaluaciones auditivas similares en todos los pacientes con CF con alta exposición a AG. El análisis genético puede ayudar a explicar la dicotomía encontrada en cuanto a la respuesta a AG.