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D C Collins

Publications and source records attributed to D C Collins.

At least 19 recordsLinked to original sources
PubMed2005

The effect of active immunization against adrenocorticotropic hormone on cortisol, beta-endorphin, vocalization, and growth in pigs.

Because the poor growth performance of intensively housed pigs is associated with increased circulating glucocorticoid concentrations, we investigated the effects of glucocorticoid suppression by inducing a humoral immune response to ACTH on physiological and production variables in growing pigs. Grower pigs (28.6 +/- 0.9 kg) were immunized with amino acids 1 through 24 of ACTH conjugated to ovalbumin and suspended in diethylaminoethyl (DEAE) dextran-adjuvant or adjuvant alone (control) on d 1, 28, and 56. The ACTH-specific antibody titers generated suppressed increases in cortisol concentrations on d 63 in response to an acute stressor (P = 0.002; control = 71 +/- 8.2 ng/mL; ACTH-immune = 43 +/- 4.9 ng/mL) without altering basal concentrations. Plasma beta-endorphin concentrations were also increased (P < 0.001) on d 63 (control = 18 +/- 2.1 ng/mL; ACTH-immune = 63 +/- 7.3 ng/mL), presumably because of a release from negative feedback on the expression of proopiomelanocortin in pituitary corticotropes. Immunization against ACTH did not alter ADG (P = 0.120; control = 1,077 +/- 25; ACTH-immune = 1,143 +/- 25 g) or ADFI (P = 0.64; control = 2,719 +/- 42; ACTH-immune = 2,749 +/- 42 g) and did not modify behavior (P = 0.681) assessed by measuring vocalization in response to acute restraint. In summary, suppression of stress-induced cortisol responses through ACTH immunization increased beta-endorphin concentrations, but it did not modify ADG, ADFI, or restraint vocalization score in growing pigs.

Adrenocorticotropic Hormone↗
PubMed2001

Developments in ion mobility spectrometry-mass spectrometry.

Ion mobility spectrometry (IMS) has been used for over 30 years as a sensitive detector of organic compounds. The following is a brief review of IMS and its principles with an emphasis on its usage when coupled to mass spectrometry. Since its inception, IMS has been interfaced with quadrupole, time-of-flight, and Fourier-transform ion cyclotron resonance mass spectrometry. These hybrid instruments have been employed for the analysis of a variety of target analytes, including biomolecules, explosives, chemical warfare degradation products, and illicit drugs.

Journal Article↗
PubMed2001

Electrospray ionization gas-phase electrophoresis under ambient conditions and it's potential or high-speed separations.

A moderately high resolution nanoelectrospray ionization gas-phase electrophoresis instrument was constructed and evaluated for simple high-speed separations of several groups of compounds. The insertion of a plate containing a 1.6 cm diameter exit orifice, 2.5 cm from the location of electrospray, allowed ions to be created and desolvated under ambient conditions with minimal solvent contamination to the drift tube. Ion separation selectivity is discussed and shown to be slightly altered by changing the drift gas flow rate. Issues of using gas-phase electrophoresis as a high-speed separation technique are discussed. Gas-phase electrophoresis-spectra of selected benzodiazepines, triazine herbicides, and simple combinatorial chemistry libraries are demonstrated.

Alprazolam↗
PubMed2001

Site-directed mutagenesis studies of rat steroid 5alpha-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions.

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5alpha-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K(m)(NADPH) and an approximately fourfold decrease in the V(max) with no change in K(m)(T). The mutation G185W resulted in a fivefold decrease in K(m)(NADPH) and an eightfold decrease in V(max), with no change in K(m)(T), whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V(max), an approximately 20-fold increase in K(m)(T) and no alteration in K(m)(NADPH), whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2001

The safe journeys of an enterprising school: negotiating landscapes of opportunity and risk.

In this paper, we discuss recent efforts to improve the safety of children travelling to and from New Zealand's largest primary school. The results of a travel survey completed by parents and pupils are reported, together with our recommendations for reducing congestion at the school gate and promoting healthy alternatives to car travel. Reflecting on this research, we find that market-oriented education reforms have provided schools with strong incentives for increasing their rolls--a course of action which may endanger pupils' well-being. At the same time, they have provided some schools with opportunities for resisting the present urban (dis)order and promoting community health.

Accidents, Traffic↗
PubMed2000

New Zealand children's health camps: therapeutic landscapes meet the contract state.

This paper surveys the history and current status of children's health camps in New Zealand, and places these sites within the theoretical context of therapeutic landscapes. The first health camp was established in 1919, and the seven current camps provide respite, education and health care for approximately 4000 children each year. We analyse the health-place relations inherent in the health camp concept and suggest that the 'therapeutic landscape' idea developed by Gesler provides a useful framework to explain the development of camps as sites for enhancing child and family welfare. Specifically, we contend that changing understandings of health and children have been closely linked with changing perceptions of what is therapeutic about the camps. Survey data demonstrate that contemporary restructuring of the welfare state has recast the role of health camps and placed them in a precarious position in terms of both financial viability and public acceptability. We conclude that the current status of health camps is ambiguous given the pressures of deinstitutionalisation philosophies and the regulatory environment of formal contracts between funders and providers.

Child↗
PubMed1999

Analysis of the steroid binding domain of rat steroid 5alpha-reductase (isozyme-1): the steroid D-ring binding domain of 5alpha-reductase.

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5alpha-androst an-17beta-carboxamide is an effective probe of rat steroid 5alpha-reductase (isozyme-1) (5alphaR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5alphaR-1 activity were ultraviolet (UV)-photolyzed with [3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5alphaR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55-56 min. Rechromatography of this fraction using a modified gradient (elution 54-55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15-18 of the 5alphaR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an approximately 12-fold increase in the Km for testosterone, whereas the Km for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1999

Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino acid residues.

Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1999

Metabolism of dihydrotestosterone in human liver: importance of 3alpha- and 3beta-hydroxysteroid dehydrogenase.

This study compared the enzyme activity of 3alpha-hydroxysteroid dehydrogenase (3alphaHSD) and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the human liver. 3AlphaHSD was found in both microsomal and cytosolic liver fractions. Contrary to that in rat liver, microsomal 3alphaHSD activity was 12-fold higher than cytosolic 3alphaHSD activity, and 3alphaHSD was not inhibited by indomethacin (10 micromol/L). The rate of 5alpha-dihydrotestosterone (DHT) reduction to 5alpha-androstane-3alpha,17beta-diol (3alphaDIOL) by 3alphaHSD was 2 times higher than the rate of 3alphaDIOL oxidation to DHT. 3BetaHSD was present primarily in the microsomal fraction of the human liver, and the rate of DHT reduction to 5alpha-androstane-3beta,17beta-diol (3betaDIOL) by 3betaHSD was 3 times higher than the rate of 3betaHSD oxidation to DHT. When 3alphaHSD and 3betaHSD activities were compared, the rate of DHT reduction by 3betaHSD was 3-fold lower than the rate of DHT reduction by 3alphaHSD. No sex or age differences were found in either 3alphaHSD or 3betaHSD activity. As the activity of DHT-metabolizing enzymes is not sex dependent, the sex differences in plasma levels of 3alphaDIOL glucuronide probably reflect differences in DHT production rather than in DHT metabolism. Comparison of the activities of 3alphaHSD, 3betaHSD, and androgen UDP-glucuronyl transferase suggests that the major pathway of DHT metabolism in human liver involves 3alphaHSD reduction in the liver, followed by subsequent glucuronidation and clearance via the kidney.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Uptake of suramin by human microvascular endothelial cells.

We have demonstrated for the first time that suramin is taken up by human dermal microvascular endothelial (HMEC-1) cells by an active process involving the caveolae system. The uptake of suramin was time-dependent and reduced by more than 90% when incubated in the presence of albumin or at 4 degrees C. Suramin uptake was also inhibited when incubated in the presence of filipin and digitonin, both potent cholesterol-binding agents, but not in the presence of probenecid. The [3H]suramin taken up by the HMEC-1 cells was located primarily within the nucleus, followed by the cytoplasmic fraction. The presence of suramin in these cellular compartments suggests that this drug may act through intracellular mechanisms.

Antineoplastic Agents↗
PubMed1998

Antiangiogenic and antiproliferative activity of suramin analogues.

The purpose of this study was to test the ability of 70 polyanionic analogues of suramin to inhibit angiogenesis. The ID50, the dose that produced 50% inhibition of angiogenesis, was determined for suramin and each of the analogues by measuring the ability of various amounts to inhibit angiogenesis in vivo in the chick egg chorioallantoic membrane (CAM) assay. Of the 70 analogues, 11 had antiangiogenic activities similar to suramin and an additional 7 were significantly more potent than suramin. All seven of these analogues were from the naphthalenetrisulfonic acid group and contained large urea groups. The benzene sulfonic and disulfonic acid analogues were less active inhibitors of angiogenesis than the naphthalenetrisulfonic acid analogues. Replacement of the naphthalenetrisulfonic acid groups by aliphatic carboxylic acids or benzoic acid gave analogues with very little antiangiogenic activity. In subsequent experiments, the antiproliferative activity of selected analogues on basic FGF (bFGF)-stimulated growth of immortalized human microvascular endothelial cells in vitro was determined. Analogues that inhibited angiogenesis to a greater extent than suramin in the CAM assay generally showed a greater antiproliferative effect on bFGF-induced growth of human microvascular endothelial cells. These results suggest that some of the polyanionic analogues may be potent therapeutic agents for cancers and angiogenesis-dependent diseases.

Animals↗
PubMed1997

Expression of rat steroid 5 alpha-reductase (isozyme-1) in Spodoptera frugiperda, SF21, insect cells: expression of rat steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Preimplantation hormonal differences between the conception and non-conception menstrual cycles of 32 normal women.

We compared daily urinary concentrations of oestrogen and progesterone metabolites in paired menstrual cycles (conception and non-conception) from 32 women. Volunteers with no known fertility problems were enrolled in the study at the time they began trying to become pregnant. They collected first-morning urine specimens and kept daily records of menstrual bleeding and sexual intercourse for 6 months or until they became clinically pregnant. Intercourse in non-conception cycles was close to the time of ovulation so that failure to conceive was caused by factors other than poorly timed intercourse. Compared with non-conception cycles, conception cycles had a steeper early luteal rise in progesterone and higher mid-luteal oestrogen and progesterone concentrations. These hormonal characteristics may be markers of better quality cycles, but because all these differences were in the luteal phase, we cannot rule out the possibility that the preimplantation embryo had stimulated early increases in steroid production. We propose an analysis strategy that could help support or refute the importance of preimplantation embryonic signalling, but our small sample size limits our own conclusions about this mechanism.

Adult↗
PubMed1996

Intravenous vs. intraprostatic administration of N-methyl-N-nitrosourea to induce prostate cancer in rats.

To develop an improved model of human prostate cancer, 16-wk-old Wistar rats were treated orally for 18 days with the antiandrogen, flutamide (50 mg/kg body weight [BW]/day), followed by 3 days of s.c. testosterone (100 mg/kg BW). There were the only treatments the control animals received (Group 1, n = 10). On the day after the third testosterone injection, N-methyl-N-nitorsourea (MNU) was administered via the tail vein at a dose of 50 mg/kg BW (Groups 2, n = 40 and 3, n = 20); in some rats, a second dose was delivered by the same route 22 wk later (Group 3). A smaller dose of MNU (15 mg/kg BW) was administered intraprostatically (Group 4, n = 20) to a fourth group. In Groups 2, 3, and 4, silastic capsules containing testosterone were implanted s.c. approximately every 6 wk beginning 1 wk post-MNU. Accessory sex gland tumors arose in MNU-treated rats in Group 2 (12/40, 30%). Group 3 (8/20, 40%), and Group 4 (8/20, 40%); 90% were macroscopic (25/28). There were no neoplasms in these organs in the control rats (Group 1, 0/10). These accessory sex gland neoplasms were adenocarcinomas or undifferentiated carcinomas which appeared to be derived from the prostate based on location and histological characteristics, although the size and spread of some of the tumors precluded definitive localization of the tissue of origin. The incidence of these neoplasms was similar in rats given a single dose of MNU intraprostatically or two doses of MNU i.v., but the animals treated intraprostatically maintained higher body weights and developed fewer extraneous tumors. The average (+/- SD) latent period for clinical or postmortem detection of prostate neoplasia after MNU was shortest in the rats given two i.v. doses (39 +/- 3 wk) compared with the single i.v. dose (45 +/- 6 wk) or an intraprostatic dose (56 +/- 7 wk). In 57% of the cases (16/28), the prostate tumors metastasized to distant sites. An activating point mutation was detected in codon 12 of the Ki-ras oncogene in the MNU-induced primary prostate tumors (8/10 examined), and metastases arising from these prostate tumors (2/3) but was absent in normal prostate tissue (0/6). This study demonstrates that two systemic doses of MNU increase the incidence and decrease the latency of prostate neoplasms compared with a single dose, and that a single dose of MNU injected intraprostatically induces prostate adenocarcinoma without many of the other tumors and weight loss typically found after i.v. administration.

Adenocarcinoma↗
PubMed1996

Photoaffinity labeling of rat steroid 5 alpha-reductase (isozyme-1) by a benzophenone derivative of a 4-methyl-4-azasteroid.

[1,2-3H]N-4(Benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha-androstane-17 beta-carboxamide ([3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5 alpha-reductase isozyme-1 (5 alpha R-1). Reversible binding of the probe to 5 alpha R-1 in microsomal preparations yielded a reversible dissociation constant (Kd) of -3 nM, whereas inhibition experiments indicated that the probe had a 50% inhibition concentration of 4.4 nM and was a competitive inhibitor of the enzyme (Ki approximately 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5 alpha R-I photolyzed with [3H]4MABP in the presence of NADPH showed that the radioactivity was incorporated into a single protein band with a mass of 26 kDa (apparent molecular weight of 5 alpha R-1). UV photolysis was accompanied by an irreversible loss in enzyme activity, consistent with its covalent modification. Increasing the time of UV irradiation and concentration of [3H]4MABP indicated that the half-life and apparent Kd for its photo insertion were approximately 3 min and 7.5 nM, respectively. Photolysis in the presence of a 20-fold excess of N,N-diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5 alpha R-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP+. The results indicate that [3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5 alpha R-1.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].

Adenine↗