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PubMed2005

Vitamin D binding protein, bone status and body composition in community-dwelling elderly men.

The vitamin D binding protein (DBP) is the major carrier protein for vitamin D metabolites in plasma. Polymorphisms in DBP have been described to be associated with an increased bone fracture risk and diabetes. The present study investigates the influence of both phenotypic and (TAAA)(n)-Alu repeat DBP-polymorphism and DBP-concentration on bone mineral density, body composition, bone turnover- and metabolic markers in a cohort of ambulatory elderly men. We included 211 men (>70 years) in this study. Bone mineral density (BMD) was determined by dual energy X-ray absorptiometry. Bone turnover was assessed by measurement of serum osteocalcin, serum and urinary C-terminal telopeptides of type I collagen and urinary deoxypyridinoline, together with 25(OH)-vitamin D and 1,25(OH)(2)-vitamin D concentrations. DBP-phenotypes were determined electrophoretically and the (TAAA)(n)-Alu repeat polymorphism was determined by polymerase chain reaction. Body composition was estimated using bioelectrical impedance analysis, together with handgrip and arm strength, fasting serum glucose and leptin concentrations. No differences in BMD or bone turnover markers among DBP-phenotypes or (TAAA)(n)-genotypes were observed in this study. Serum 25(OH)-vitamin D was comparable among DBP-variants and did not relate to DBP-concentrations, whereas 1,25(OH)(2)-vitamin D was different among DBP-phenotypes and was correlated positively with DBP-concentrations. DBP-concentrations related positively to body mass index, fat mass, leptin and glucose concentration. The correlation with leptin remained significant after correction for fat mass. Fasting glucose concentrations were different among DBP-phenotypes, whereas no difference was observed between (TAAA)(n)-genotypes. In conclusion, serum 1,25(OH)(2)-vitamin D concentrations are codetermined by DBP-phenotypes and DBP-concentrations. No major effect of DBP-polymorphism was demonstrated on BMD, bone turnover markers or body composition.

Absorptiometry, Photon↗
PubMed1990

Uptake and degradation of vitamin D binding protein and vitamin D binding protein-actin complex in vivo in the rat.

We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver.

Actins↗
PubMed1989

The vitamin D-binding protein, alpha-fetoprotein, albumin multigene family: detection of transcripts in multiple tissues.

The serum vitamin D-binding protein (DBP), a member of a multigene family that includes alpha-fetoprotein (alpha FP) and albumin (ALB), is expressed at high levels in the adult liver. Although several studies have demonstrated the presence of DBP on the cell surface of immunocytes and cytotrophoblasts, evidence of extra-hepatic DBP synthesis remains inconclusive. Using Northern blot analysis of RNA from multiple rat tissues, the DBP transcripts were present in the expected high levels in the adult liver. In addition, DBP mRNA was found in the adult kidney, testis, abdominal fat, and 18-day fetal yolk sac. The concentrations of DBP mRNA in these tissues is 100-1000-fold less than in the liver. Polymerase chain reaction amplification (PCR) of DBP, alpha FP, and ALB cDNAs, reverse transcribed from a wide variety of rat tissue RNA samples, was performed to examine their respective tissue-specific patterns of gene transcripts. The tissues in which DBP gene transcripts were noted by Northern analysis were paralleled at 20 cycles of PCR with liver much greater than kidney, yolk sac, testis, and abdominal fat. Similarly at 20 cycles, alpha FP-amplified fragments were detected in the yolk sac much greater than brain, testes, uterus, liver, and placenta; and ALB-amplified fragments in liver much greater than testis, uterus, placenta, and yolk sac. Unexpectedly, at 32 cycles of PCR, DBP, alpha FP, and ALB mRNAs could be detected at very low levels in all tissues examined. To examine the nature of the DBP mRNA from these nonhepatic sources, a Sprague-Dawley kidney cDNA library was screened, and a single cDNA recombinant was identified. This clone contained a full length DBP cDNA with 14 nucleotide and 3 predicted amino acid differences from the Fischer liver DBP cDNA reported previously. We conclude that the kidney and liver DBP mRNAs are transcription products of the same DBP locus and that DBP, alpha FP, and ALB genes display different spectra of tissue distribution. The unexpected finding that all three spliced RNA transcripts could be detected in at least trace quantities in all tissues studied suggests a leaky level of transcription by this multigene family or their transcription at low levels by an unidentified stromal cell common to all organs studied.

Amino Acid Sequence↗
PubMed2005

Comparison of fixed-dose combinations of telmisartan/hydrochlorothiazide 40/12.5 mg and 80/12.5 mg and a fixed-dose combination of losartan/hydrochlorothiazide 50/12.5 mg in mild to moderate essential hypertension: pooled analysis of two multicenter, prospective, randomized, open-label, blinded-end point (PROBE) trials.

BACKGROUND: High incidences of cardiovascular events coincide with a surge in blood pressure (BP) that occurs in the early morning hours at the time of arousal. Thus, control of BP at this time of day, using oral fixed-dose combinations (FDCs) as required, is important in reducing cardiovascular risk in hypertensive patients. OBJECTIVE: The aim of this analysis was to compare the antihypertensive efficacy in the early morning hours and tolerability of oral FDCs of telmisartan/hydrochlorothiazide (HCTZ) (40/12.5 mg [T40/H12.5] and 80/12.5 mg [T80/H12.5]) versus a low-dose FDC of losartan 50 mg/HCTZ 12.5 mg (L50/H12.5). METHODS: Data from 2 similarly designed prospective, randomized, open-label, blinded-end point (PROBE) studies were pooled and analyzed. The studies were conducted at 72 centers across the United States, and 70 centers in Canada, Europe (9 countries), and the Philippines. Adult male and female patients with mild to moderate essential hypertension (24-hour mean ambulatory diastolic BP [DBP], > or =85 mm Hg; seated cuff DBP, 90-109 mm Hg) were enrolled. Patients were randomly assigned to receive T40/H12.5, L50/H12.5, or T80/H12.5, QD (morning) for 6 weeks. Antihypertensive efficacy was assessed using 24-hour ambulatory BP monitoring (ABPM) and cuff sphygmomanometry at trough, performed at baseline and on completion of active treatment. The primary end point was the reduction from baseline in mean ambulatory DBP over the last 6 hours of the dosing interval. Secondary end points included other ABPM- and clinic-derived changes in DBP and systolic BP (SBP), and control and response rates (SBP response defined as 24-hour mean SBP <130 mm Hg and/or reduction from baseline > or =10 mm Hg; DBP response defined as 24-hour mean DBP <85 mm Hg or reduction from baseline > or =10 mm Hg; DBP control defined as 24-hour mean DBP <85 mm Hg). Tolerability was assessed using patient interview, spontaneous reporting, and clinical evaluation. RESULTS: A total of 1402 patients were enrolled(876 men, 525 women; mean [SD] age, 53.1 [9.9] years) (T40/H12.5, n = 517; L50/H12.5, n = 518; and T80/H12.5, n = 367). With T40/H12.5, the mean reduction in last-6-hour mean ambulatory DBP was 1.8 mm Hg greater compared with that achieved with L50/H12.5 (-11.3 [0.4] vs -9.4 [0.4] mm Hg; P < 0.001), and with T80/H12.5, the mean reduction was 2.6 mm Hg greater compared with that achieved with L50/H12.5 (-12.0 [0.4] vs -9.4 [0.4] mm Hg; P < 0.001). Analysis of secondary end points found that greater BP reduction occurred with T40/H12.5 and T80/H12.5 compared with L50/H12.5. ABPM SBP control and response rates were similar between the 3 groups, but the ABPM DBP control and response rates were significantly higher with T80/H12.5 compared with L50/H12.5 (46.6% vs 34.0% [P < 0.002] and 69.4% vs 55.0% [P < 0.001], respectively). Clinic SBP and DBP control and response rates were higher with T40/H12.5 and T80/H12.5 compared with L50/H12.5 (SBP response, 80.4% and 80.8% vs 68.5% [both, P < 0.001]; DBP response, 66.1% and 67.4% vs 54.4% [both, P < 0.001]; DBP control, 56.5% and 56.4% vs 44.1% [both, P < 0.001] ). The 2 most commonly recorded adverse events (AEs) were headache (T40/H12.5, 2.9%; L50/H12.5, 3.3%; and T80/H12.5, 3.0%) and dizziness (1.2%, 2.1%, and 3.0%, respectively). Most AEs were mild to moderate. CONCLUSIONS: The results of this pooled analysis of2 PROBE studies in adult patients with mild to moderate essential hypertension suggest that T40/H12.5 and T80/H12.5 conferred greater DBP and SBP control compared with low-dose L50/H12.5, including during the last 6 hours of the dosing interval. All 3 treatments were well tolerated.

Angiotensin II Type 1 Receptor Blockers↗
PubMed2002

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein.

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.

Actins↗
PubMed1984

Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli.

Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected. This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP. One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction. Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition. The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination. Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP. The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations. All phenotypic effects attributed to strain 6204 appear to be due to a single mutation. Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min. Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp. The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls. Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls. Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective. These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis. To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP. These strains had growth properties that were identical with those of strain 6204. Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride. Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.

Butylene Glycols↗
PubMed1990

Cell surface vitamin D-binding protein (GC-globulin) is acquired from plasma.

Vitamin D-binding protein (DBP) is an abundant plasma protein. The observation of immunodetectable, cell-associated DBP on peripheral blood mononuclear cells and placental cytotrophoblasts had presented the question of the origin, function, and precise subcellular localization of cell-associated DBP. Using anti-human DBP F(ab')2 with fluorescence-activated cytometric analysis and immunogold electron microscopy, we detected DBP on the plasmalemma of U937 cells, a monoblastic, histiocytic cell line grown in media supplemented with fetal calf serum (FCS). DBP was then removed from FCS by actin affinity chromatography followed by anti-DBP immunoaffinity chromatography. U937 cells in this DBP-free medium exhibited nearly identical growth rates to cells grown in medium containing native FCS. However, in contrast to cells grown with native FCS, those grown for seven to eight generations with DBP-free FCS exhibited less cell-surface DBP as quantified by fluorescence-activated cytometric analysis (73% decrease) and immunoelectron microscopy (88% decrease). DBP mRNA could not be detected in U937 cells, placental tissues, freshly prepared resting and stimulated B and T lymphocytes, or lymphocyte-derived cell lines by Northern analysis. In addition, using the sensitive reverse transcriptase/polymerase chain reaction assay no DBP fragments were detectable in U937 cells. We conclude that U937 cell-associated DBP is exogenously derived from plasma and is located on the plasmalemma. Based upon this conclusion, we postulate that specific binding sites for DBP may exist on the plasma membranes of certain cell types.

Blotting, Northern↗
PubMed1992

Receptor-mediated uptake and processing of vitamin D-binding protein in human B-lymphoid cells.

Vitamin D-binding protein (DBP), a member of a multigene family including alpha-fetoprotein (AFP) and albumin, is a serum glycoprotein that reversibly binds and transports vitamin D and its metabolites to target cells. In this work, we demonstrate that normal and malignant human B-lymphocytes specifically bind and internalize DBP. Radioiodinated DBP (125I-DBP) was used to follow the uptake of the protein by Raji cells, a human pre-B-lymphoma cell line. Time course studies of DBP uptake by these cells exhibited a saturable profile at both 4 and 37 degrees C. The binding saturation curve obtained by incubating Raji cells at 4 degrees C with different concentrations (1.5 nM to 1.5 microM) of 125I-DBP showed two saturation plateaus; Scatchard analysis showed the presence of two groups of receptor sites with a Kd1 of 2.04 x 10(-7) M (n1 = 42,161 +/- 4,336 sites/cell) and a Kd2 of 1.01 x 10(-6) M (n2 = 198,000 +/- 48,000 sites/cell). After incubation of Raji cells at 37 degrees C with both fluorescein isothiocyanate (FITC) and horseradish peroxidase conjugates, DBP was internalized and could be localized in the cytoplasm. DBP-horseradish peroxidase conjugates were used to follow the uptake and to determine the endocytic pathway of the protein in Raji cells. The initial steps, contrary to those observed for AFP, did not apparently involve coated pits and vesicles. Small vesicles (approximately 50-60 nm) with electron-dense DBP-horseradish peroxidase reaction products were observed that could fuse with large endosomes. These endosomes appeared dispersed in the cytoplasm with some preferential localization in the Golgi centrosphere region. Pulse-chase experiments showed that only 10% of the uptaken protein was released in a nondegraded form. Accordingly, most DBP molecules accumulated in endosomes should be degraded in lysosomes, instead of being recycled back to the surface, as in the case of AFP. Contrary to malignant B-cells (Raji), the uptake ability for DBP of normal quiescent B-lymphocytes was very low. Specific binding and internalization of DBP-FITC by these cells were observed following mitogen-induced activation. Significant values of uptake were obtained at 37 degrees C after 72 h of incubation in the presence of pokeweed mitogen. The binding of DBP-FITC was partially inhibited in the presence of an excess of unlabeled protein. Taken together, the actual results suggest that DBP receptors are constitutively expressed by malignant B-cells and in a transitory form by normal B-lymphocytes undergoing mitogen-induced activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding.

The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus DNA replication.

Adenoviridae↗
PubMed2006

Bioremediation of environmental endocrine disruptor di-n-butyl phthalate ester by Rhodococcus ruber.

In this study DBP-degradation strain CQ0301 was isolated from rubbish landfill soil. According to the biophysical, biochemical characteristics and analysis of 16S rRNA, the strain was identified as Rhodococcus ruber. Three new protein bands could be fractioned after DBP-inducing, which were suspected to participate the process of DBP-degrading. Catechol was suspected to be an intermediate product of DBP and cleaving the benzene ring was catalyzed by catechol 1,2-dioxygenase, because a highly activity of catechol 1,2-dioxygenase could be detected after DBP-inducing. The results of this study also showed the optimal pH value, optimal temperature which influenced the degradation rate in soil: pH 7.0-8.0, 30-35 degrees C. Kinetics of degradation reaction had been performed at different initial concentration and different time. Analyzed with SPSS10.0 software, the DBP degradation can be described as the same exponential model when the initial DBP concentration was lower than 50 mg/kg. The kinetics equation was lnC=-0.1332t + A, with the degradation half-life of DBP in soil (5.20 d). Inoculating CQ0301 could relieve DBP content in plant. We also found that adding nutrient materials into soil was useful for decreasing the DBP content in plant. In summary, we isolated a bacterium capable of degrading DBP and decreasing DBP content in plant. We also explored the mechanism of biodegradation and characterized the environmental factors influencing the degradation process in contaminated soil. Based on this work, we hope that these findings can provide some information for applying of bioremediation of DBP contaminated soil.

Biodegradation, Environmental↗
PubMed1980

Isolation and partial characterization of two immunologically similar vitamin D-binding proteins in rat serum.

Two immunologically similar, probably identical, binding proteins for vitamin D and its metabolites (DBP1 and DBP2) were isolated separately from rat serum after approximately 180-fold purification by novel procedures using Blue Sepharose CL-6B chromatography. The freshly purified DBP1 and DBP2 each showed a single band of protein on polyacrylamide gel electrophoresis, and had alpha-mobility, although DBP1 moved slightly faster than DBP2. DBP1 and DBP2 had the same molecular weight, which was estimated as approximately 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric points of DBP1 and DBP2 were estimated as 4.9 and 5.0, respectively, from the results of isoelectric focusing experiments. DBP1 and DBP2 both appeared to have one binding site for 25-hydroxyvitamin D3 per molecule of protein, with apparently similar association constants at 4 degrees C of 5--7 x 10(9) M-1. The amino acid compositions of DBP1 and DBP2 were also determined and compared. A monospecific antiserum against rat DBP2 was prepared in a rabbit and was used for immunological studies of rat DBP. On double immunodiffusion, anti-DBP2 antiserum produced precipitin lines of complete reaction-of-identity against the purified DBP1, the purified DBP2, and rat whole serum. There was no immunological cross-reactivity between rat DBP and sera from man, dog, and rabbit, but mouse serum showed a pattern of partial identity with rat DBP. When rat serum samples were analyzed by immunoelectrophoresis using anti-DBP antiserum, three patterns of precipitin line were observed: a pattern showing the existence of only DBP1, designated as DBP 1-1; a pattern showing the existence of only DBP2, designated as DBP 2-2; and a pattern showing the existence of both DBP1 and DBP2, designated as DBP 2-1. Using single radial immunodiffusion assay for rat serum DBP, the mean (+/- S.D.) serum DBP concentrations were found to be 461 +/- 59 microgram/ml in adult male rats and 328 +/- 16 microgram/ml in adult female rats, and the difference was significant (p < 0.001). In molar terms, DBPs are present in normal rat serum in large excess relative to vitamin D and its metabolites, and most of the serum DBP, therefore, circulates as apo-DBP, not containing a bound molecule of vitamin D or of its metabolites. The immunoprecipitation studies of DBP in rat serum showed that DBPs were common main transport proteins for naturally occurring vitamin D and its metabolites, and that DBP played some, but not a principal, role in the transport of synthetic 1 alpha-hydroxyvitamin D3.

Amino Acids↗
PubMed1984

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.

Adenoviruses, Human↗
PubMed1995

Blood pressure and mortality among men with prior myocardial infarction. Multiple Risk Factor Intervention Trial Research Group.

BACKGROUND: The purpose of the present study was to describe the relation between blood pressure (systolic [SBP] and diastolic [DBP]) and death from coronary heart disease (CHD) and all causes for men with a history of myocardial infarction (MI). METHODS AND RESULTS: The study cohort consisted of men aged 35 to 57 years screened for the Multiple Risk Factor Intervention Trial (MRFIT) in 1973 through 1975 and followed for survival for an average of 16 years through 1990. There were 5362 men who reported prior hospitalization for a heart attack of at least 2 weeks' duration at the initial screening of MRFIT. There was a J-shaped relation between SBP and DBP with both CHD and all-cause mortality during the first 2 years of follow-up in older (age, 45 to 57 years) men only. Risk nadirs for SBP were 152 and 145 mm Hg, respectively, for CHD death and all-cause mortality; corresponding DBP risk nadirs were 94 and 90 mm Hg. After the first 2 years, there was a positive association between SBP and death from CHD and all causes. By 15 years, cumulative CHD mortality percentages for men with screening SBP < 120, 120 to 139, 140 to 159, and > or = 160 mm Hg were 19.7%, 21.3%, 27.5%, and 32.0%, respectively. When deaths only after year 2 were considered, although the linear DBP coefficient was significant, the quadratic term for DBP was no longer significant (P > .05). However, the relation still appeared J-shaped as cumulative mortality for those with DBP < 70, 70 to 79, 80 to 89, 90 to 99, and > or = 100 mm Hg was 24.3%, 20.8%, 21.1%, 25.5%, and 29.7%, respectively. When the joint relation of SBP and DBP was considered, there were no survival differences among the four cohorts (SBP > or = 140 and DBP < 80, SBP > or = 140 and DBP > or = 80, SBP < or = 140 and DBP < 80, and SBP < or = 140 and DBP > or = 80) during the first 2 years. After 2 years, both CHD and all-cause mortality rates were approximately 40% higher for participants with SBP > or = 140 mm Hg versus < 140 mm Hg regardless of DBP level (< 80 or > or = 80 mm Hg). CONCLUSIONS: In this large cohort of men with prior MI, the association of SBP and DBP with CHD and all-cause mortality varied over the 16-year follow-up period. During early follow-up, in older men only, J- or U-shaped relations were evident. However, after 2 years, these same relations had become positive and graded. Given the substantial excess mortality risk in this cohort associated with high blood pressure, particularly SBP, efforts to gradually lower blood pressure should receive high priority among hypertensive men with prior MI.

Adult↗
PubMed2003

Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism.

In utero exposure to di(n-butyl) phthalate (DBP) leads to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists. DBP demonstrates no affinity for the androgen receptor, but rather leads to diminished testosterone production by the fetal testis. The purpose of this study was to determine the onset and reversibility of DBP effects on the fetal testis and to determine at a functional level the points in the cholesterol transport and steroidogenesis pathways affected by DBP. Starting at gestational day (gd) 12, pregnant rats were gavaged daily with 500 mg/kg DBP or corn oil control. Significant decreases in testosterone production and mRNA expression of scavenger receptor B1, P450(SCC), steroidogenic acute regulatory protein, and cytochrome p450c17 were observed as early as gd 17. Testosterone, mRNA, and protein levels remained low 24 h after withdrawal of DBP treatment but increased 48 h after cessation of DBP exposure. In another experiment, pregnant dams were treated with DBP until gd 19, with the start of DBP treatment moved 1 d later into gestation for each treatment group, with the final group dosed only on gd 19. Significant decreases in testosterone, mRNA expression, and protein expression were evident as early as 3 h after treatment with DBP, with full repression apparent 24 h after treatment. Using a testis explant system, we determined that DBP treatment led to diminished transport of cholesterol across the mitochondrial membrane as well as diminished function at each point in the testosterone biosynthesis pathway except 17 beta-hydroxysteroid dehydrogenase. The transcriptional repression caused by DBP does not appear to be mediated via interference with steroidogenic factor-1 as determined by reporter assays. We conclude that high-dose DBP exposure leads to rapid and reversible diminution of the expression of several proteins required for cholesterol transport and steroidogenesis in the fetal testis, resulting in decreased testosterone synthesis and consequent male reproductive maldevelopment.

Animals↗
PubMed2001

Elastase controls the binding of the vitamin D-binding protein (Gc-globulin) to neutrophils: a potential role in the regulation of C5a co-chemotactic activity.

The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37 degrees C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.

Cell Membrane↗
PubMed1999

Initial characterization of the vitamin D binding protein (Gc-globulin) binding site on the neutrophil plasma membrane: evidence for a chondroitin sulfate proteoglycan.

The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.

Binding Sites↗
PubMed2005

Biodegradation characteristics of environmental endocrine disruptor di-n-butyl phthalate.

OBJECTIVE: The biodegradation characteristics of di-n-butyl phthalate (DBP), an environmental endocrine disruptor, were studied by the method of dominant bacteria and immobilized microorganisms. METHODS: Taking DBP as the only carbon source to acclimatize the collected activated sludge, the concentration of DBP increased progressively in the process of acclimatization. Plate streaking was used to separate 1 strain of the degradation dominant bacteria after acclimatization. Better conditions to degrade DBP by the bacterium could be obtained through orthogonal experiments and the bacterium was identified. Then the acclimated activated sludge was made to immobilize the microorganism using polyvinyl alcohol as entrapment agent. The immobilized microorganism degraded DBP at different conditions. RESULTS: The appropriate conditions to degrade DBP by the dominant bacteria were: degradation time, 32 h; DBP concentration, 200 mg/L; rate of shaking incubator, 100 r/min; pH, 7 and temperature, 30 degrees C. DBP could be degraded by more than 95% under such conditions. The bacteria were identified as Pseudomonas. The proliferated immobilized microorganisms degraded DBP more effectively and more adapted to temperature and pH than the free acclimated activated sludge. CONCLUSION: One strain of DBP degradation dominant bacteria was separated from the acclimatized activated sludge. It could grow with DBP as the only carbon source and energy, and degraded DBP effectively. After having been immobilized and proliferated, the dominant bacteria could keep a higher biological activity and degrade DBP more effectively than activated sludge.

Biodegradation, Environmental↗
PubMed1984

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene.

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.

Adenoviruses, Human↗