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PubMed2002

Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system.

Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin D-binding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 A resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to "clamp" onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-A(2) intermolecular interface involve a mixture of hydrophobic, electrostatic, and solvent-mediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin-DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.

Actins↗
PubMed2001

Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate.

Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.

Androgen Antagonists↗
PubMed2005

Subchronic exposure to low concentrations of di-n-butyl phthalate disrupts spermatogenesis in Xenopus laevis frogs.

Due to its widespread use and production, di-n-butyl phthalate (DBP) has become an environmental contaminant. It has been detected in a variety of environmental strata worldwide, including air, water, and soil. Also, monobutyl phthalate, the major metabolite of DBP, has been detected in a variety of human matrices. As a proven endocrine disruptive compound, DBP may contribute to global amphibian declines at much lower concentrations than tested thus far. We evaluated the effects of low concentrations of DBP on spermatogenesis in Xenopus laevis, the African clawed frog. Xenopus tadpoles were exposed to 0, 0.1, 0.5, 1.0, 5.0, or 10.0 ppm DBP, beginning at sexual differentiation (Nieuwkoop and Faber stage 52; 3 weeks of age) and continuing until 100% of controls metamorphosed (stage 66; 8 weeks of age). Upon necropsy at 33 weeks, 4-6% of DBP-treated frogs had only one testis, and 2-4% had retained oviducts. In all DBP treatment groups, seminiferous tubule diameter and the average number of germ cell nests per tubule were lower, and the number of tubules with no germ cells was significantly higher (p < 0.05). The percent of secondary spermatogonial cell nests significantly decreased (p < 0.05) in 1.0, 5.0, and 10.0 ppm groups. Several lesions occurred in DBP-exposed testes including denudation of germ cells, vacuolization of Sertoli cell cytoplasm, thickening of lamina propria of seminiferous tubules, and focal lymphocytic infiltration. Entire sections of testes containing almost exclusively mature spermatozoa were found in 1.0, 5.0, and 10.0 ppm DBP-exposed testes, indicating impairment of spermiation. Testicular hypoplasia and seminiferous tubular dysgenesis were also evident in DBP-treated frogs. Thus, subchronic exposure to low concentrations of DBP impairs spermatogenesis in Xenopus laevis frogs.

Animals↗
PubMed2002

Variability within alpha- and beta-adrenoreceptor genes as a predictor of cardiovascular function at rest and in response to mental challenge.

OBJECTIVES: To investigate the association between polymorphic variation in alpha- and beta-adrenoreceptor genes and cardiovascular activity at rest and in response to psychological challenge in a sample in which the heritability of these cardiovascular phenotypes may be established. METHODS: Several common polymorphisms were characterized within ADRA1B (alpha1B), ADRA2A (alpha2A), ADRB1 (beta1) and ADRB2 (beta2) and examined in relation to heart rate (HR) and systolic (SBP) and diastolic (DBP) blood pressure, both at rest and in response to stress. Participants were 309 European-American, young adult men and women (including 101 monozygotic and 44 dizygotic twin pairs). RESULTS: In the full sample, participants carrying any G allele at base pair (bp) 1165 in ADRB1 exhibited elevated resting SBP and DBP and a larger DBP response to mental challenge compared to homozygotes for the C allele (P < 0.04). An AA genotype at bp 145 in ADRB1 was also associated with higher resting SBP and DBP than AG or GG genotypes (P < 0.03). At bp 46 in ADRB2, GG homozygotes had higher resting DBP than subjects possessing any A allele (P < 0.05). For the same polymorphism, however, AG heterozygotes showed lower SBP than both AA and GG homozygotes (P < 0.05). In a subsample of genetically unrelated individuals, ADRB1 (1165) continued to predict resting SBP, DBP and DBP response to stress (P < 0.03), while ADRB2 (46) was associated with resting SBP (P < 0.04) but not DBP. Finally, the degree of allele sharing at ADRB1 (1165) also predicted variability in SBP and DBP at rest among dizygotic twin pairs (P < 0.04). CONCLUSIONS: These results indicate that some polymorphic variation within adrenoreceptor genes contributes to interindividual variability in resting SBP and DBP and in DBP response to mental challenge.

Adult↗
PubMed1985

Transformation of human 143 tk- cells with plasmids containing the gene encoding the adenovirus DNA-binding protein.

Human cell lines that contain and express the gene encoding the adenovirus type 5 DNA-binding protein (Ad5 DBP) are very useful for the isolation of adenovirus mutants with an altered DBP. In order to obtain these cells, human 143 tk- cells were transfected, using the calcium phosphate technique, with plasmids containing the Ad5 DBP gene and the herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker. Characterization of several tk+ transformants revealed that these cells did contain the HSV tk gene, but in none of these cells could Ad5 DBP DNA sequences be detected. However, when 143 tk- cells were co-transfected with a plasmid containing the Ad5 DBP gene and another plasmid carrying early region E1, integration of the Ad5 DBP gene in chromosomal DNA could be detected. Integration of Ad5 DNA sequences was also observed when transfection was performed with plasmids containing the Ad5 DBP gene and the long terminal repeat of Moloney murine leukaemia virus. By employing a radioimmunoassay it could be shown that DBP-related proteins were synthesized in two of the cell lines containing the Ad5 DBP gene. Since both cell lines support the growth of the temperature-sensitive viral DBP mutant, H5ts125, at the non-permissive temperature, the DBP-related proteins expressed in these cells must be functional.

Adenoviruses, Human↗
PubMed1977

Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells.

High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-beta-d-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Ad11 (group B)-infected, and weakly with Ad12 and Ad18 (group A)-infected KB cells (treated with 1-beta-d-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.

Adenoviruses, Human↗
PubMed1999

Differential impact of systolic and diastolic blood pressure level on JNC-VI staging. Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure.

The sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure classifies blood pressure into stages on the basis of both systolic (SBP) and diastolic (DBP) blood pressure levels. When a disparity exists between SBP and DBP stages, patients are classified into the higher stage ("up-staged"). We evaluated the effect of disparate levels of SBP and DBP on blood pressure staging and eligibility for therapy. We examined 4962 Framingham Heart Study subjects between 1990 and 1995 and determined blood pressure stages on the basis of SBP alone, DBP alone, or both. After the exclusion of subjects on antihypertensive therapy (n=1306), 3656 subjects (mean age 58+/-13 years; 55% women) were eligible. In this sample, 64.6% of subjects had congruent stages of SBP and DBP, 31.6% were up-staged on the basis of SBP, and 3.8% on the basis of DBP; thus, SBP alone correctly classified JNC-VI stage in approximately 96% (64.6%+31.6%) of the subjects. Among subjects >60 years of age, SBP alone correctly classified 99% of subjects; in those </=60 years old, SBP alone correctly classified 95%. Of 1488 subjects with high-normal blood pressure or hypertension, who were potentially eligible for drug therapy, 13.0% had congruent elevations of SBP and DBP, 77.7% were up-staged on the basis of SBP, and 9.3% were up-staged on the basis of DBP; SBP alone correctly classified 91%, whereas DBP alone correctly classified only 22%. SBP elevation out of proportion to DBP is common in middle-aged and older persons. SBP appears to play a greater role in the determination of JNC-VI blood pressure stage and eligibility for therapy. Given these results, combined with evidence from hypertension treatment trials, future guidelines might consider a greater role for SBP than for DBP in determining the presence of hypertension, risk of cardiovascular events, eligibility for therapy, and benefits of treatment.

Adult↗
PubMed1984

Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease.

We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration, and the level of vitamin D-binding protein (DBP) in 24 normal subjects, 17 pregnant subjects, and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus, the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl, respectively, P less than 0.001), whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml, respectively). In contrast, in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%, P less than 0.001), the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml, P less than 0.001). When data from all three groups were combined, there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels, (b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels, and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions.

Adult↗
PubMed1987

Vitamin D binding protein sequesters monomeric actin in the circulation of the rat.

Plasma vitamin D binding protein (DBP) may scavenge actin released during cell lysis. We examined the plasma disappearance and tissue appearance of 125I-DBP, 125I-G-actin, and the DBP-G-actin complex after their intravenous administration to rats. The plasma disappearance of DBP and DBP-actin were indistinguishable, with rapid initial (t1/2 = 2.6 h) and slower second (t1/2 = 7 h) slopes. After 125I-G-actin (nanomole) injection, plasma disappearance paralleled that of DBP and DBP-actin. All injected actin was associated with DBP, without evidence of free actin, actin-gelsolin complexes or actin oligomers. Tissue appearances of 125I-apo-DBP (apo) or holo-DBP were similar, with highest accumulations in perfused liver, kidney, and skeletal muscle. Although more complex phenomena (plasma entry of F-actin and intracellular actin binding proteins) would occur in vivo after cell lysis, our results suggest a role for DBP in the sequestration and disposition of actin monomers in the circulation.

Actins↗
PubMed1987

Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae.

Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with [35S]methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated.

Actins↗
PubMed2006

Antihypertensive effect of peptides obtained from Enterococcus faecalis-fermented milk in rats.

Previous studies have demonstrated that milk fermented with Enterococcus faecalis decreases the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of spontaneously hypertensive rats. In this study, we evaluated the antihypertensive activity of the following peptide sequences: LHLPLP, LHLPLPL, LVYPFPGPIPNSLPQNIPP, VLGPVRGPFP, and VRGPFPIIV. These peptides isolated from E. faecalis-fermented milk showed in vitro angiotensin I-converting enzyme-inhibitory activity. Because the most potent angiotensin I-converting enzyme-inhibitory sequences were LHLPLP and LVYPFPGPIPNSLPQ-NIPP, we administered different doses of these peptides to spontaneously hypertensive rats. High doses of the remaining sequences were also administered to these animals. Water served as a negative control and captopril as a positive control. All products were administered orally. The SBP and DBP were measured before administration and also at 2, 4, 6, 8, and 24 h after administration. Before administration of the different products, spontaneously hypertensive rats showed SBP and DBP values of 218 +/- 2.5 and 157 +/- 5.9 mmHg, respectively (n = 30). The sequences LHLPLP, LVYPF-PGPIPNSLPQNIPP, VLGPVRGPFP, and VRGPFPIIV caused clear and significant decreases in SBP, DBP, or both in the animals. In particular, the antihypertensive effect could be clearly established when 2 or 3 mg/kg of LHLPLP was administered. These 2 doses of LHLPLP showed similar antihypertensive properties. Four hours after administration of captopril or the highest doses of the different peptides, the decreases in the SBP and the DBP (mmHg) were as follows: captopril (SBP = 52 +/- 5.8, DBP = 38.8 +/- 3.8), 3 mg/kg of LHLPLP (SBP = 25.3 +/- 8.2, DBP = 29.5 +/- 7.6), 6 mg/kg of LVYPFPGPIP-NSLPQNIPP (SBP = 14.9 +/- 3.7, DBP = 8.7 +/- 4.4), 10 mg/kg of LHLPLPL (SBP = 7.7 +/- 4.1, DBP = 9.4 +/- 3.1), 10 mg/kg of VLGPVRGPFP (SBP = 16.2 +/- 5.8, DBP = 21.64 +/- 3.2), and 10 mg/kg of VRGPFPIIV (SBP = 16.05 +/- 2.74, DBP = 9.19 +/- 3.49). The results obtained suggest that the sequences LHLPLP, LVYPFPGPIPNSLPQ-NIPP, VLGPVRGPFP, and VRGPFPIIV could be responsible, at least in part, for the antihypertensive properties described for E. faecalis-fermented milk.

Animals↗
PubMed2004

Platelet-derived thrombospondin-1 is necessary for the vitamin D-binding protein (Gc-globulin) to function as a chemotactic cofactor for C5a.

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D-binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56-kDa plasma protein that binds and transports several diverse ligands. The objective of this study was to investigate the mechanisms by which DBP functions as a chemotactic cofactor for C5a using neutrophils and U937 cells transfected with the C5aR (U937-C5aR cells). The results demonstrate that U937-C5aR cells show C5a chemotactic enhancement only to DBP in serum, but, unlike mature neutrophils, this cell line cannot respond to DBP in plasma or to purified DBP. Analysis by SDS-PAGE and isoelectric focusing revealed no structural difference between DBP in serum compared with DBP in plasma. However, plasma supplemented with either serum, DBP-depleted serum, or activated platelet releasate provides a required factor and permits DBP to function as a chemotactic cofactor for C5a. Fractionation of activated platelet releasate revealed that the additional factor possessed the properties of thrombospondin-1 (TSP-1). Finally, purified TSP-1 alone could reproduce the effect of serum or platelet releasate, whereas Abs to TSP-1 could block these effects. These results provide clear evidence that TSP-1 is needed for DBP to function as a chemotactic cofactor for C5a.

Adjuvants, Immunologic↗
PubMed1992

Renin predicts diastolic blood pressure response to nonpharmacologic and pharmacologic therapy.

OBJECTIVE: Plasma renin activity was measured at baseline and 6 months in a trial of nonpharmacologic therapy of mild hypertension to determine whether plasma renin activity predicts the diastolic blood pressure (DBP) response to nonpharmacologic therapy. DESIGN, SETTING, AND PARTICIPANTS: Randomized controlled trial of volunteers from the general community with mild hypertension (DBP between 90 and 100 mm Hg), off all antihypertensive therapy at baseline, treated in special research clinics (n = 593). INTERVENTIONS: Subjects were randomly assigned to usual, weight loss, or low sodium/high potassium diet and then randomly assigned to receive placebo, chlorthalidone, or atenolol. MAIN OUTCOME MEASURES: Renin was analyzed as plasma renin activity and as a renin index (logarithm of 24-hour urinary sodium excretion times logarithm of plasma renin activity) (593 patients at baseline and 6 months) to correct for varied sodium intakes. The DBP was measured using the random zero device. RESULTS: Change in DBP at 6 months could be predicted from baseline plasma renin activity or renin index. The DBP was decreased after 6 months of therapy by 2 mm Hg for each unit increase in baseline plasma renin activity and by 0.16 mm Hg for each unit increase in baseline renin index. Patients in the highest renin index quartile had a greater DBP response to atenolol therapy, and patients in the lowest renin index quartile had a greater DBP response to chlorthalidone therapy. Weight loss diet achieved a greater reduction in DBP in patients with higher baseline renin index and had an additive effect on DBP response in both of the drug groups. Patients on a weight loss diet receiving placebo in the highest baseline renin index quartile had a reduction in DBP of 12.4 mm Hg, compared with 4.4 mm Hg in the lowest baseline renin index quartile (P = .009). A low sodium/high potassium diet had a lesser effect than a weight loss diet on pharmacologic therapy. As with the weight loss diet, patients on a low sodium/high potassium diet in the highest baseline renin index quartile had a greater reduction in DBP than patients in the lowest baseline renin index quartile. CONCLUSIONS: These data suggest a significant relationship between baseline levels of plasma renin index and the likelihood of success of nonpharmacologic treatment of hypertension.

Adult↗
PubMed1993

Localization and quantitation of the vitamin D binding protein (Gc-globulin) in human neutrophils.

Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell-free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.

Blotting, Western↗
PubMed2000

Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols.

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ¿25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ¿1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ¿(14)C25-OH-D(3)-11-BE and ¿(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ¿(14)C25-OH-D(3)-6-BE and ¿(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ¿(14)C25-OH-D(3)-3-BE or ¿(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.

Acetates↗
PubMed1996

Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.

Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.

Actins↗
PubMed1998

ATP-independent DNA unwinding by the adenovirus single-stranded DNA binding protein requires a flexible DNA binding loop.

The adenovirus DNA binding protein (DBP) binds cooperatively to single-stranded (ss) DNA and stimulates both initiation and elongation of DNA replication. DBP forms protein filaments via a C-terminal arm that hooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Another conserved part of DBP forms an unstructured flexible loop that is probably directly involved in contacting DNA. By making appropriate deletion mutants that do not distort the overall DBP structure, the influence of the C-terminal arm and the flexible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface plasmon resonance we show that both parts of the protein are required for high affinity binding. Deletion of the C-terminal arm leads to an extremely labile DBP-ssDNA complex indicating the importance of multimerization. The flexible loop is also required for optimal stability of the DBP-ssDNA complex, providing additional evidence that this region forms part of the ssDNA-binding surface of DBP. Both deletion mutants are still able to stimulate initiation of DNA replication but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Surprisingly, mixtures containing both mutants do stimulate elongation. Mixing the purified mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These results provide evidence that the C-terminal arm and the flexible loop have distinct functions in unwinding during replication. We propose the following model for ATP-independent DNA unwinding by DBP. Multimerization via the C-terminal arm is required for the formation of a protein filament that saturates the displaced strand. A high affinity of a DBP monomer for ssDNA and subsequent local destabilization of the replication fork requires the flexible loop.

Adenosine Triphosphate↗
PubMed1985

Pulse rate, blood pressure and body composition in black adolescents: the Philadelphia Blood Pressure Project.

Patterns of pulse rate, blood pressure (BP), and body size and composition are investigated in a black urban sample of over 600 adolescents (aged 12-17) in Philadelphia as part of the Philadelphia Blood Pressure Project. Supine and seated pulse rates decline after the age of 13 in both males and females, and females have significantly higher pulse rates than males, ranging from 1.6 to 4.7 beats/30 sec higher depending upon chronological age and position. Seated pulse rates are consistently higher than supine in both sexes ranging from 1.3 to 2.9 beats/30 sec depending upon chronological age. Pulse rate and both systolic blood pressure (SBP) and diastolic phase IV blood pressure (DBP) show a positive association throughout adolescence for females (r = 0.16 SBP, r = 0.24 DBP) but not for males (r = -0.06 SBP, r = 0.06 DBP). Females in the highest diastolic IV blood pressure stratum (greater than 85%) maintain higher pulse rates throughout adolescence, while males with the highest DBP have higher pulse rates in early adolescence (ages 12-14) but lower pulse rates in later adolescence (ages 16 and 17). Females with the highest DBP also show consistently higher measures of relative fatness (triceps skinfolds) throughout adolescence. Males with the highest DBP have greater triceps skinfolds in early adolescence and lower triceps in later adolescence, a pattern similar to that shown for pulse rate. For DBP, triceps is significantly and positively associated with pulse rate in the upper DBP percentile group (p less than 0.05). Thus, females with the highest blood pressure during adolescence are characterized by higher pulse rate and greater fatness. Males show this same pattern in early adolescence; whereas, in contrast, the older adolescent males in our sample with the highest DBP are characterized by lower pulse rates, relatively decreased fatness, and increased muscularity. Our findings suggest that, at least in some older black male adolescents, a higher DBP may reflect a decrease in cardiac output as indicated by a lower pulse rate, or an increase in peripheral vascular resistance, possibly associated with relatively decreased fatness and increased muscle mass for size.

Adolescent↗