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PubMed1992

Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication.

The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.

Adenoviruses, Human↗
PubMed1983

Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein.

Two hybridoma cell lines were obtained, A1 and B6, which produced monoclonal antibodies reacting with the 44,000-MW C-terminal domain of the adenovirus type 5 DNA-binding protein (DBP). Clone A1 antibodies reacted with the native form of the DBP, but failed to recognize this protein after denaturation (by exposure to sodium dodecylsulfate, or production of the DBP at 39.5 degrees by H5ts107, a temperature-sensitive DBP mutant). Clone B6 antibodies bound to both the native and denatured forms of the DBP. Immunofluorescent staining of wild-type-virus infected cells revealed the DBP located in discrete nuclear patches. A1 and B6 antibodies detected this patched localization of the DBP in nuclei of H5ts107-infected cells grown at 32 degrees. However, at the nonpermissive temperature of 39.5 degrees, A1 antibodies failed to detect the DBP, and B6 antibodies gave a uniform nuclear fluorescent distribution of the DBP. Thus the nuclear pattern of localization for the DBP synthesized by H5ts107 was temperature dependent in this mutant.

Adenoviruses, Human↗
PubMed1992

Polyunsaturated fatty acids decrease the apparent affinity of vitamin D metabolites for human vitamin D-binding protein.

The affinity of purified human vitamin D-binding protein from serum (DBP) for 25-hydroxyvitamin D3 (25-OHD3) and 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was measured in the presence of free fatty acids (FFA), cholesterol, prostaglandins and several drugs. Mono- and polyunsaturated fatty acids markedly decreased the affinity of both 25-OHD3 and 1,25-(OH)2D3 for DBP, whereas saturated fatty acids (stearic and arachidic acid), cholesterol, cholesterol esters, retinol, retinoic acid and prostaglandins (A1 and E1) did not affect the apparent affinity. Several chemicals known to decrease the binding of thyroxine to its plasma-binding protein did not affect the affinity of DBP. The apparent affinity of DBP for both 25-OHD3 and 1,25-(OH)2D3 decreased 2.4- to 4.6-fold in the presence of 36 microM of linoleic or arachidonic acid, respectively. Only a molar ratio of FFA:DBP higher than 10,000 was able to decrease the binding of 25-OHD3 to DBP by 20%. Much smaller ratio's of FFA:DBP (25 for arachidonic and 45 for oleic acid), however, decreased the binding of 1,25-(OH)2D3 to DBP. These latter ratio's are well within the physiological range. The addition of human albumin in a physiological albumin:DBP molar ratio did not impair the inhibitory effect of linoleic acid on the binding of [3H]25-OHD3 to DBP. The binding and bioavailability of vitamin D metabolites thus might be altered by mono- and polyunsaturated but not by saturated fatty acids.

Albumins↗
PubMed2004

Efficacy and safety of delapril plus manidipine compared with enalapril plus hydrochlorothiazide in mild to moderate essential hypertension: results of a randomized trial.

BACKGROUND: The use of combination therapy is required to achieve blood pressure targets in 40% to 75% of patients with hypertension. There have been few studies comparing the efficacy and tolerability of the new fixed combination of the angiotensin-converting enzyme (ACE) inhibitor delapril 30 mg and the calcium channel antagonist manidipine 10 mg with those of a standard combination of another ACE inhibitor and a diuretic. OBJECTIVE: The aim of this study was to compare the antihypertensive efficacy and tolerability of delapril 30 mg given alone or with manidipine 10 mg with those of enalapril 20 mg given alone or with hydrochlorothiazide (HCTZ) 12.5 mg in patients with mild to moderate essential hypertension. METHODS: This was a multicenter, active-controlled, parallel-group trial. After an initial 2-week placebo run-in period, patients aged 18 to 75 years with diastolic blood pressure (DBP) > or =90 and < or =109 mm Hg were randomized in a 2:1 ratio to receive delapril or enalapril for 8 weeks. After the initial 8 weeks, nonresponders (DBP > or =85 mm Hg) received an additional 8 weeks of treatment with a fixed combination of delapril + manidipine or enalapril + HCTZ; patients whose DBP was normalized continued their initial monotherapy through the end of the study. The primary efficacy variable was the change in sitting DBP at the end of treatment. Secondary efficacy variables were the percentage of patients whose DBP was normalized (DBP Z:85 mm Hg) and the percentage of responders (> or =10-mm Hg reduction in DBP or DBP <85 mm Hg). RESULTS: One hundred sixty patients (84 men, 76 women) were randomized to receive delapril (n = 106) or enalapril (n = 54). After 16 weeks of treatment, the mean (SD) reduction in DBP was similar with the 2 treatments (delapril, -14 [8] mm Hg; enalapril, -15 [8] mm Hg). In the delapril and enalapril groups, DBP was normalized in a respective 55 (51.9%) and 29 (53.7%) patients, and 77 (72.6%) and 38 (70.4%) were responders; there was no significant difference between groups. Tolerability was also similar in both groups--10 (9.4%) patients in the delapril group and 5 (9.3%) in the enalapril group experienced adverse events that were judged related to treatment. CONCLUSIONS: The results of this study suggest that delapril alone or combined with manidipine is well tolerated and as effective as enalapril alone or combined with HCTZ in lowering blood pressure in patients with mild to moderate essential hypertension.

Adolescent↗
PubMed2005

Association between human atrial natriuretic peptide Val7Met polymorphism and baseline blood pressure, plasma trough irbesartan concentrations, and the antihypertensive efficacy of irbesartan in rural Chinese patients with essential hypertension.

BACKGROUND: Individual variations in the pharmacokinetics and pharmacodynamics of antihypertensive drugs are influenced by genetic and environmental factors. The ANP gene, which encodes the precursor of atrial natriuretic peptide (ANP), is among the candidate genes for genetic susceptibility to hypertension. OBJECTIVE: This study examined the relationship between ANP Val7Met polymorphism (Single Nucleotide Polymorphism database ID: rs5063) and baseline blood pressure (BP), plasma trough irbesartan concentrations, and the antihypertensive efficacy of irbesartan in rural Chinese patients with essential hypertension. METHODS: Patients with essential hypertension who had taken no antihypertensive medications within 4 weeks of study initiation received oral irbesartan 150 mg/d for 4 weeks. Genotyping was performed for all patients. BP was measured before dosing on the 1st and 28th days of treatment. Plasma irbesartan concentrations were measured using high-performance liquid chromatography with fluorescent detection. Antihypertensive efficacy was defined as attainment of a diastolic BP (DBP) <90 mm Hg (DBP analysis), a systolic BP (SBP) <140 mm Hg (SBP analysis), and a DBP <90 mm Hg and SBP <140 mm Hg (DBP and SBP analysis). RESULTS: The study included 756 patients, 621 with the Val/Val genotype and 135 with the Val/Met+Met/Met genotypes. There were no significant differences in age, body mass index, sex, education level, occupation, alcohol consumption, or smoking status between the 2 groups. Patients with the Val/Met+Met/Met genotypes had a significantly lower mean baseline DBP compared with those with the Val/Val genotype (adjusted regression coefficient [SE]: -2.5 [1.0] mm Hg; P = 0.012) and significantly lower mean steady-state plasma trough irbesartan concentrations (adjusted regression coefficient: -12.6 [4.1]; P = 0.002). No significant association was found between antihypertensive efficacy and Val7Met polymorphism in the overall population, but in an analysis by baseline DBP status, patients with the Val/Met+Met/Met genotype a baseline DBP > or =100 mm Hg had significantly smaller reductions in DBP (adjusted regression coefficient: -5.7 [1.4] mm Hg; P < 0.001) and SBP compared with those with the Val/Val genotype and a baseline DBP > or =100 mm Hg (adjusted regression coefficient: -9.8 [2.9] mm Hg; P < 0.001). CONCLUSION: The findings of this study suggest that in these rural Chinese patients with essential hypertension, ANP Val7Met polymorphism may be a genetic marker for baseline DBP, plasma irbesartan concentrations, and the antihypertensive efficacy of short-term irbesartan therapy.

Adult↗
PubMed2005

Selective inhibition of the C5a chemotactic cofactor function of the vitamin D binding protein by 1,25(OH)2 vitamin D3.

The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D3, and G-actin plus 25(OH)D3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)2D3) completely eliminated the chemotactic cofactor function of DBP. Dose-response curves demonstrated that as little as 1pM 1,25(OH)2D3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)2D3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B4 was not altered by 1,25(OH)2D3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)2D3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)2D3.

Actins↗
PubMed1998

Further evaluation of developmental toxicity of di-n-butyl phthalate following administration during late pregnancy in rats.

The objective of this study was to further evaluate the developmental toxicity of di-n-butyl phthalate (DBP) administered during the second half of pregnancy. Pregnant rats were fed a diet containing DBP at a dose of 0 (control), 0.5, 1.0 or 2.0% ad libitum on days 11-21 of pregnancy. Average daily intakes of DBP were 331, 555 and 661 mg/kg for the 0.5, 1.0 and 2.0% groups, respectively. No significant changes induced by DBP were detected in the incidence of postimplantation loss and numbers of live fetuses and of resorptions and dead fetuses. The weights of male and female fetuses at 2.0% DBP were significantly decreased. The incidences of fetuses with cleft palate and fetuses with fusion of the sternebrae at 2.0% DBP and fetuses with undescended testes at 1.0 and 2.0% DBP were significantly increased. There were significant decreases in the anogenital distance (AGD) of male fetuses in the 1.0 and 2.0% DBP groups, also. AGD of female fetuses in the DBP-treated groups was comparable to that in the control group. It was concluded that DBP administered during the second half of pregnancy produced adverse effects on the reproductive development in male fetuses.

Abnormalities, Drug-Induced↗
PubMed2000

Critical period for adverse effects on development of reproductive system in male offspring of rats given di-n-butyl phthalate during late pregnancy.

The objective of this study was to determine the susceptible days for the adverse effects of di-n-butyl phthalate (DBP) on development of reproductive system in male offspring following maternal administration on successive 3-day period during late pregnancy. Pregnant rats were given DBP by gastric intubation at 1000 or 1500 mg/kg on days 12-14 or 18-20 of pregnancy or at 500, 1000 or 1500 mg/kg on days 15-17 of pregnancy. A significant decrease in the maternal body weight gain and/or food consumption was found in the DBP-treated groups regardless of the days on which DBP at 1000 and 1500 mg/kg was given. A significant increase in the number of resorptions per litter was found in the groups given DBP at 1500 mg/kg on days 12-14 and 15-17 of pregnancy. The weights of male and female fetuses were significantly decreased in the groups given DBP at 1000 and 1500 mg/kg on days 12-14 and 18-20 and at 1500 mg/kg on days 15-17. A significant increase in the incidence of fetuses with undescended testes was found at 1500 mg/kg on days 12-14 and at all doses on days 15-17. A significant decrease in the anogenital distance (AGD) of male fetuses was observed in the groups treated with DBP regardless of the days of treatment. The AGD/body weight ratio in male fetuses was significantly reduced in the groups given DBP on days 15-17, but neither on days 12-14 nor 18-20. The AGD of female fetuses in the DBP-treated groups was comparable to that in the control group. It was concluded that period of days 15-17 of pregnancy was the most susceptible for DBP-induced undescended testes and decreased AGD in male offspring.

Animals↗
PubMed1998

Hypertension in young patients after renal transplantation: ambulatory blood pressure monitoring versus casual blood pressure.

The results of ambulatory blood pressure monitoring (ABPM) in children after kidney transplant were analyzed to ascertain any alteration in circadian BP profile, degree of hypertension, and efficacy of therapy. The data were also compared with casual BP data and left ventricular mass index (LVMI). We have examined 30 patients (17 male, 13 female), mean age 16.1+/-3.6 years after kidney transplant. All patients were receiving triple immune-suppressive therapy and 20 of them were also taking antihypertensive therapy. They underwent clinical examination with measurement of BP at rest, echocardiogram mono-2D, and ABPM. The following ABPM parameters were recorded: systolic (S) and diastolic (D) BP at rest; mean 24-h SBP and DBP; mean daytime SBP and DBP; mean nighttime SBP and DBP; nocturnal fall in SBP and DBP; and mean daytime and nighttime heart rate (HR). The patients were divided in two groups. Group A consisted of 20 patients taking antihypertensive treatment; group B consisted of 10 patients not taking antihypertensive treatment. Casual and ABPM data for the two groups were compared using the Student t test for unpaired data. Blood pressure at rest and LVMI were not statistically different between the two patient groups. The ABPM data showed statistical differences between the two groups for mean 24-h SBP and DBP, daytime and nighttime SBP, nighttime DBP, fall in nocturnal DBP, and nighttime HR. Mean 24-h SBP and DBP, mean daytime SBP and DBP, and mean nighttime SBP and DBP were significantly correlated to LVMI (respectively, P = .009, P = .005, P = .008, P = .007, P = .05, and P = .01). Twenty-four-hour ABPM was more useful in the diagnosis and management of hypertension than was casual BP at rest.

Adolescent↗
PubMed2005

Hyperventilation and amplified blood pressure response: is there a link?

Based on prior studies, the hypothesis that hyperventilation (HV) may have a pressor effect and play a causal role in hypertension has been suggested. The objective of this study was to correlate HV with blood pressure (BP)-change during a postural challenge. Consecutive subjects referred for evaluation of syncope, dizziness, chronic fatigue syndrome (CFS), fibromyalgia, or non-CFS fatigue were assessed with a 10-min supine 30-min head-up tilt test combined with capnography. We selected for analysis the records of patients aged 17-70 years, not taking vasoactive medications, having sitting systolic BP (SBP) < 140 mmHg, sitting diastolic BP (DBP) < 90 mmHg, and who completed 30 min of tilt. HV was diagnosed when end-tidal pressure of CO2 < 30 mmHg was recorded consecutively for > or = 10 min. Postural hypertension (PHT) was diagnosed when DBP on tilt > or = 90 mmHg was recorded consecutively for > or = 10 min. DBP-change was computed as (median DBP on tilt) -(median DBP supine). PHT and DBP-change were correlated with HV. A total of 320 patient charts were reviewed. PHT was present in 30 cases. The mean DBP-change in patients with PHT was +9.9 mmHg (s.d. 5.8), with three patients manifesting HV. Of the remaining 290 patients, 56 had HV, their mean DBP-change was -0.3 mmHg (s.d. 7.2). The other 234 patients without HV had a mean DBP-change +0.95 mmHg (s.d. 5.7), comparable to the DBP-change in patients with HV. In, conclusion, posturally induced HV was not associated with an increase in BP, nor was PHT associated with HV, except in a small minority of cases.

Adolescent↗
PubMed2006

Blood pressure as a predictor of cardiovascular events in the elderly: the William Hale Research Program.

This study evaluates the association between blood pressure (BP) and the risk of developing cardiovascular disease (CVD) events in the elderly. The Morton Plant Mease Foundation has followed 4,008 elderly patients >64 years of age for at least 5 years. Systolic and diastolic blood pressure (SBP and DBP) was divided into categories. Cardiovascular disease events were classified as myocardial infarction, stroke, and CVD-related deaths reported from the National Death Index. Cox proportional hazard ratios were used to assess the relationship between BP and CVD events and controlled for weight, gender, smoker, and alcohol use. Ages <75 and >or=75 years were assessed separately. After 11.1 years of follow-up, elevated SBP (P=<0.0001) is strongly associated with developing a future CVD event; the relationship is linear and graded and holds for ages above and below 75 years. The frequency of CVD events was lowest in the SBP <120 mm Hg group. In subjects <75 years of age, DBP elevations were not a significant risk factor for CVD events. (relative risk (RR): DBP 70 to <80 mm Hg=0.92; DBP 80 to <90 mm Hg=0.88; DBP >or=90 mm Hg=1.02.) With subjects >or=75 years of age, a DBP between 80 and 90 is associated with the lowest significant risk for CVD (RR: DBP 70 to <80 mm Hg=0.74; DBP 80 to <90 mm Hg=0.59; DBP >or=90=0.71). In conclusion, these findings support the Joint National Committee on Hypertension recommendations for SBP in the elderly. Further studies are warranted to identify optimal DBP for the elderly at various ages.

Aged↗
PubMed1998

Sources of variation in the determination of distal blood pressure measured using the strain gauge technique.

Using the determination of distal blood pressure (DBP) measured using the strain gauge technique as an example of a routine clinical physiological investigation involving many different observers (laboratory technicians), the present study was carried out to assess (1) the influence of the number of observers and the number of analyses made by each observer on the precision of a definitive value; and (2) the minimal difference between two determinations to detect a real change. A total of 45 patients participated in the study. They were all referred for DBP determination on suspicion of arterial peripheral vascular disease. In 30 of the patients, the DBP curves were read twice, with a 5-week interval, by 10 laboratory technicians. The results were analysed using the variance component model. The remaining 15 patients had their DBP determined twice on two different days with an interval of 1-3 days and the total day-to-day variation (SDdiff) of DBP was determined. The inter- and intraobserver variations were, respectively, 5.7 and 4.9 mmHg at ankle level and 3.5 and 2.7 mmHg at toe level. The index values as related to systolic pressure were somewhat lower. The mean day-to-day variation was 11 mmHg at ankle level and 10 mmHg at toe level, thereby giving a minimal significant difference between two DBP determinations of 22 mmHg at ankle and 20 mmHg at toe level. To decrease the value of SD (standard deviation) on a definitive determination of DBP and index values, it was slightly more effective if the value was based on two observers performing one independent DBP curve reading than if one observer made one or two DBP curve readings. The reduction in SDdiff was greatest at ankle level. The extent of the Sddiff decrease was greatest when two different observers made a single DBP reading each at both determinations compared with one different observer making two readings at each determination. Surprisingly, about half of the maximum reduction in the SDdiff was achieved just by increasing the number of observers from one to two. We have found variance component analyses to be a suitable method for determining intra- and interobserver variation when several different observers take part in a routine laboratory investigation. It may be applied to other laboratory methods such as renography, isotope cardiography and myocardial perfusion single-photon emission computerized tomography (SPECT) scintigraphy, in which the final result may be affected by individual judgement during processing.

Aged↗
PubMed1999

D-site binding protein transactivation requires the proline- and acid-rich domain and involves the coactivator p300.

The D-site binding protein (DBP) is a member of the proline- and acid-rich (PAR) domain subfamily of basic/leucine zipper proteins and is involved in transcriptional regulation in the liver. Deletion analysis of the DBP protein was carried out in an effort to define the function of the conserved PAR domain. Internal deletions of the protein, i.e. removing portions of the PAR domain, resulted in a substantial loss in transactivation of a high affinity DBP reporter construct when assayed in Hep G2 cells. These same sequences conferred significant transactivation to GAL4 DNA binding domain fusion proteins, indicating that this region acts as part of an independent activation domain comprised of sequences in both the amino terminus and in the PAR domain of DBP. The coexpression of full-length expression constructs for both DBP and hepatic leukemia factor resulted in a dramatic increase in activation mediated by the GAL4-DBP fusion proteins, suggesting the involvement of a regulated coactivator in this process. DBP transactivation appears to be a p300-dependent process, as a 12 S E1A expression construct disrupted DBP-mediated transactivation, and a p300 expression vector, but not a CREB binding protein vector, was able to restore DBP transactivation. These results suggest that the PAR domain is required for DBP activation, which occurs through a regulated, p300-dependent process.

Amino Acid Sequence↗
PubMed2004

Antigenic drift in the ligand domain of Plasmodium vivax duffy binding protein confers resistance to inhibitory antibodies.

Interaction of the Duffy binding protein (DBP) with its erythrocyte receptor is critical for maintaining Plasmodium vivax blood-stage infections, making DBP an appealing vaccine candidate. The cysteine-rich region II is the ligand domain of DBP and a target of vaccine development. Interestingly, most of the allelic diversity observed in DBP is due to the high rate of nonsynonymous polymorphisms in this critical domain for receptor recognition. Similar to the hypervariability in influenza hemagglutinin, this pattern of polymorphisms in the DBP ligand domain suggests that this variation is a mechanism to evade antibody neutralization. To evaluate the role that dbp allelic diversity plays in strain-specific immunity, we examined the ability of an anti-Sal1 DBP serum to inhibit the erythrocyte-binding function of variant dbp alleles expressed on COS cells. We observed that the PNG-7.18 allele was significantly less sensitive to immune inhibition of its erythrocyte-binding activity than were the Sal1 and PNG-27.16 alleles. This result suggested that the unique polymorphisms of resistant PNG-7.18 were part of a protective epitope on the DBP ligand. To confirm this, Sal1 was converted to the refractory phenotype by introduction of 3 polymorphisms unique to PNG-7.18, via site-directed mutagenesis. The results of the present study indicate that linked polymorphisms have an additive, synergistic effect on DBP antigenic character.

Alleles↗
PubMed1997

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis.

In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.

Adenosine Triphosphate↗
PubMed2003

Human 'testicular dysgenesis syndrome': a possible model using in-utero exposure of the rat to dibutyl phthalate.

BACKGROUND: The disorders comprising human 'testicular dysgenesis syndrome' (TDS) may be increasing in incidence. TDS originates in fetal life but the mechanisms are not known, and discerning them requires an animal model. METHODS AND RESULTS: The study investigated whether male rats exposed in utero to dibutyl phthalate [DBP; 500 mg/kg on gestational days (GD) 13-21] would provide a suitable model for human TDS. DBP induced a high rate (>60%) of cryptorchidism (mainly unilateral), hypospadias, infertility and testis abnormalities, similar to those in human TDS. Cell-specific immunohistochemistry and confocal microscopy were used to track development of Sertoli [anti-Müllerian hormone (AMH), Wilm's tumour (WT-1) protein, p27(kip)], Leydig [3beta-hydroxysteroid dehydrogenase (3beta-HSD)], germ (DAZL protein) and peritubular myoid (smooth muscle actin) cells from fetal life to adulthood. In scrotal and cryptorchid testes of DBP-exposed males, areas of focal dysgenesis were found that contained Sertoli and Leydig cells, and gonocytes and partially formed testicular cords; these dysgenetic areas were associated with Leydig cell hyperplasia at all ages. Suppression ( approximately 90%) of testicular testosterone levels on GD 19 in DBP-exposed males, coincident with delayed peritubular myoid cell differentiation, may have contributed to the dysgenesis. Double immunohistochemistry using WT-1 (expressed in all Sertoli cells) and p27(kip) (expressed only in mature Sertoli cells) revealed immature Sertoli cells in dysgenetic areas. DBP-exposed animals also exhibited Sertoli cell-only (SCO) tubules, sporadically in scrotal and predominantly in cryptorchid, testes, or foci of SCO within normal tubules in scrotal testes. In all SCO areas the Sertoli cells were immature. Intratubular Leydig cells were evident in DBP-exposed animals and, where these occurred, Sertoli cells were immature and spermatogenesis was absent. Abnormal Sertoli cell-gonocyte interaction was evident at GD 19 in DBP-exposed rats coincident with appearance of multinucleated gonocytes, although these disappeared by postnatal day 10 during widespread loss of germ cells. CONCLUSIONS: Abnormal development of Sertoli cells, leading to abnormalities in other cell types, is our hypothesized explanation for the abnormal changes in DBP-exposed animals. As the testicular and other changes in DBP-exposed rats have all been reported in human TDS, DBP exposure in utero may provide a useful model for defining the cellular pathways in TDS.

Age Factors↗
PubMed2005

Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate.

The phthalate ester di(n-butyl) phthalate (DBP) causes feminization of male rats upon in utero exposure by repressing expression of genes required for testicular steroidogenesis. Previous work in our laboratory has shown that repression of gene expression and steroidogenesis in the fetal testis is apparent within a few hours of DBP exposure. The purpose of this study was to determine the precise timing of DBP-associated gene expression changes in the fetal testis using transcriptional profiling and to determine whether DBP exerts similar effects on steroidogenesis in the fetal adrenal. A DBP time-course experiment showed that testicular steroidogenesis was decreased within 1 h of DBP exposure and that this decrease preceded the repressed transcription of Star (steroidogenic acute regulatory protein); Scarb1 (scavenger receptor class B, member 1; also know as Sr-b1); Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1; also known as P450SCC); and Cyp17a1 (cytochrome P450 family 17, subfamily a, polypeptide 1; also known as Cyp17). Gene expression profiling demonstrated rapid (within 1 to 3 h) and transient induction of immediate early genes in the fetal testis after administration of DBP to the pregnant dam. There was a statistically insignificant decrease in corticosterone production by the fetal adrenal after in utero exposure to DBP from Gestation Day 12 to Gestation Day 19. The extent of steroidogenesis diminution was much less in the adrenal than in the testis (approximately 45% decrease in the adrenal versus 87% decrease in the testis) and expression of genes required for steroidogenesis in the adrenal was unaffected by DBP. Together, these studies demonstrate that DBP initiates a rapid and dynamic change in gene expression in the fetal testis that likely plays a role in the reduction in steroidogenesis that is unique to the fetal testis relative to the steroidogenically active fetal adrenal.

Adrenal Glands↗
PubMed1999

Evaluation of the potential interaction between NaCl and prostaglandin inhibition in elderly individuals with isolated systolic hypertension.

OBJECTIVE: To evaluate whether prostaglandin inhibition with the non-steroidal anti-inflammatory drug (NSAID), indomethacin (I) interacts synergistically with different doses of salt (NaCl) in elevating systolic blood pressure (SBP). DESIGN AND METHODS: This randomized, placebo-controlled, double-blind, crossover study examined the interaction between NaCl and the prostaglandin inhibitor, I in 31 healthy elderly individuals with a mean age (+/- SD) of 68.7+/-5.7 years (range 61-85 years). Participants aged more than 60 years on a 140 mmol/day NaCl dose for 6 weeks were chosen with normal blood pressure [24-h SBP <148 mm Hg, diastolic blood pressure (DBP) <85 mm Hg on the Takeda Ambulatory Blood Pressure Monitor (TABPM); n = 15] and isolated systolic hypertension (ISH), [24-h SBP >148 mm Hg, 24-h DBP <85 mm Hg on TABPM; n = 16]. Exclusion criteria included uncontrolled hypertension (SBP >220 mm Hg and/or DBP >110 mm Hg), cardiac disease, creatinine clearance <60 ml/min, dementia and recent cerebrovascular accident or secondary hypertension. A 2x2 Latin square design was structured using four treatment groups [low salt (NaCl = 90 mmol/day) + I placebo, high salt (NaCl = 240 mmol/day) + I placebo, low salt + I (25 mg three times daily) and high salt + I] for 2 weeks each, balanced and interspersed with 2 week washout periods to minimize carryover effects. Twenty-four hour SBP, DBP and heart rate were measured and summarized using a moving interval averaging technique. The mean change in 24-h SBP, DBP, heart rate, urinary Na+, K+, protein and creatinine, creatinine clearance and serum electrolytes were compared across treatments in the total cohort and in ISH and control groups separately using ANCOVA (SAS). RESULTS: In the total cohort, compared with low NaCl, chronic high NaCl increased mean SBP (5.76 mm Hg; P = 0.0002) and DBP (3.36 mm Hg; P = 0.002). Indomethacin significantly increased mean SBP (2.66 mm Hg, P = 0.015) but not DBP (0.31 mm Hg, P = 0.419). High salt and I were additive (SBPT, DBPT) but there was no interaction (P = 0.795 and P = 0.739, respectively). Additionally, chronic high NaCl increased serum Na (P = 0.0001) and 24-h urinary Na (P = 0.0001) as expected. Indomethacin significantly decreased mean heart rate (P = 0.018). The effects of NaCl and I on SBP, DBP and heart rate were not modified by age, alcohol intake, serum K+, body mass index or treatment order. In the ISH group, NaCl dose significantly elevated SBP (9.87 mm Hg; P = 0.0001) and DBP (5.26 mm Hg, P = 0.006) but did not significantly alter blood pressure in the normotensive group. Indomethacin significantly elevated SBP (P = 0.03) in normotensive individuals but had no effect on blood pressure in the ISH group. CONCLUSIONS: Chronic high salt diet elevated blood pressure more than I in the total cohort of elderly individuals. No interaction was demonstrated and their effects were additive. In the ISH group, chronic high salt diet significantly increased SBP and DBP while I failed to alter blood pressure. In the normotensive group, I, but not salt, elevated SBP. Patients with ISH are sensitive to the pressor effect of NaCl but resistant to the pressor effect of prostaglandin inhibition in contrast to elderly normotensive control individuals where the reverse was found.

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