Wildlife rehabilitation.
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Animal Welfare↗
Biomedical subjects
R D Stevens
Publications and source records attributed to R D Stevens.
Purification and characterization of the Cu,Zn SOD from Escherichia coli.
The periplasmic Cu,Zn superoxide dismutase has been purified to homogeneity by a procedure, which depended upon osmotic shock followed by two chromatographic columns. Its subunit weight, determined by electrospray ionization mass spectrometry, was found to be 15,737 +/- 1.6. The second derivative ultraviolet spectrum indicated a lack of tryptophan. The amino acid composition as well as a partial N-terminal amino acid sequence is reported. The specific activity was 3700 U/mg and the corresponding copper content was 0.77 atoms Cu/subunit. The enzyme was quite unstable and overnight dialysis against EDTA or even prolonged dialysis against neutral phosphate buffer caused partial loss of activity and of copper and visible precipitation. It is likely that some losses occurred during the isolation procedure, and if these could have been prevented the copper content would have been 1.0 Cu/subunit and the specific activity would have been 4800 U/mg. It now appears likely that gram negative bacteria will commonly be found to contain a periplasmic Cu,Zn SOD.
Amino Acid Sequence↗
Identification of five hemoglobins in B6C3F1 mice by mass spectrometry and sequence analysis.
The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two alpha-globin chains (alpha-1 and alpha-2) and at least three beta-globin chains, beta-1, beta-2 and beta-3. This is one additional alpha- and one additional beta-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the alpha-1, alpha-2, beta-2 and beta-3 chains compared to the BALB/c mouse hemoglobins (alpha, beta (minor) and beta (major)). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.
Amino Acid Sequence↗
Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.
RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.
Animals↗
Processive post-translational modification. Vitamin K-dependent carboxylation of a peptide substrate.
Mass spectrometry has been used to demonstrate that vitamin K-dependent carboxylation is a processive post-translational modification (i.e. multiple carboxylations occur during a single association between enzyme and substrate). Purified vitamin K-dependent carboxylase can carboxylate as many as 12 glutamate residues in FIXQ/S, a peptide substrate based on amino acids -18 to 41 of the human blood clotting enzyme factor IX. Mass spectrometry was used to determine the number of gamma-carboxyl groups added to FIXQ/S by the carboxylase during an in vitro reaction. Despite the fact that most substrate molecules in a reaction were uncarboxylated, almost all carboxylated FIXQ/S molecules were carboxylated many times. This observation can only be explained by two types of mechanisms. In a processive mechanism, multiple carboxylations could occur during a single substrate binding event. Alternatively, a distributive mechanism could result in the observed behavior if the initial carboxylation event results in a substrate that is additionally carboxylated far more efficiently than the uncarboxylated FIXQ/S. Kinetic experiments and arguments were used to show that the vitamin K-dependent carboxylase is not distributive but rather is one of the first well documented examples of an enzyme that catalyzes a processive post-translation modification.
Amino Acid Sequence↗
Evaluation of the use of intrauterine infusions of oxytetracycline, subcutaneous injections of fenprostalene, or a combination of both, for the treatment of retained fetal membranes in dairy cows.
Dairy cows (n = 207) that had retained fetal membranes for more than 8 hours after parturition were randomly assigned to 1 of 4 treatment groups (group 1, daily intrauterine infusion of oxytetracycline; group 2, daily intrauterine infusion of oxytetracycline and a single SC injection of fenprostalene; group 3, a single SC injection of fenprostalene; and group 4, untreated controls). Cows were monitored daily. Subsequent disease conditions were recorded, and appropriate treatments were initiated. Interval from parturition to expulsion of fetal membranes was unaffected by treatment regimen. The frequency of cows with high rectal temperatures (> or = 39.5 C) was significantly (P < 0.05) less in cows treated with oxytetracycline infusions; however, the frequency of displaced abomasum, ketosis, and mastitis was unaffected by treatment method. Treatment method appeared to influence the development of postpartum uterine pathologic conditions. Treatment with oxytetracycline and fenprostalene (group 2) increased (P < 0.05) the frequency of pyometra. Treatment with fenprostalene (groups 2 and 3) decreased the number of cows with a palpable uterine lumen detectable during per rectal palpation at 28 to 42 days after parturition. Reproductive efficiency, as determined on the basis of the interval from parturition to conception, was unaffected by treatment protocol.
Abortifacient Agents, Nonsteroidal↗
Evaluation of the effects of route of administration of cloprostenol on synchronization of estrus in diestrous dairy cattle.
Cloprostenol was administered IV or IM to diestrous dairy cows to study luteolysis and synchrony of estrus and ovulation. In Study 1, 28 Holstein cows were assigned to receive injections of cloprostenol, IV or IM, on day 8 or 10 of the estrous cycle. Differences in cloprostenol-induced luteolysis or interval to estrus, were not detected. Ultrasonography revealed that 2 cows treated IV on day 8, 1 cow treated IM on day 8, and 2 cows treated IV on day 10 did not ovulate despite evidence of an apparently normal estrus. In Study 2, 20 Holstein cows were assigned to receive cloprostenol injections, IV or IM, on day 10 of the estrous cycle. Interval to estrus was not affected by route of administration. Analysis of these data indicated that cloprostenol given IV did not alter luteolysis or improve synchrony of estrus and ovulation when compared with cloprostenol given IM.
Animals↗
Efficacy of a Propionibacterium acnes immunostimulant for treatment of chronic Staphylococcus aureus mastitis.
This study determined the efficacy of treatment of chronic Staphylococcus aureus IMI of lactating dairy cows with a biological response modifier consisting of a killed preparation of Propionibacterium acnes. Fifty-one lactating dairy cows with chronic S. aureus IMI on two commercial dairies were enrolled in a controlled, randomized field trial. Twenty-four cows received 1 ml of the immunostimulant twice weekly for 4 wk, and the remainder of the cows served as untreated controls. Quarter milk samples were collected for SCC from all cows at 3 and 6 wk after initiation of treatment and for culture at 6 wk after initiation of treatment. If samples were negative for S. aureus at 6 wk, cows were sampled again for culture 1 wk later for 2 successive d before being considered cured. For the cows treated with immunostimulant, 16.7% (36.7% of infected quarters) were cured of S. aureus IMI; for the untreated group, 11.1% of cows (32.2% of quarters) were cured. The difference was not statistically significant. Immunostimulant treatment had no effect on quarter SCC in infected quarters overall, but, in those quarters that were cured of S. aureus infection, quarter SCC of treated cows tended to be lower than SCC of control cows.
Adjuvants, Immunologic↗
Bioequivalency comparison between two gonadotropin-releasing hormone products.
The bioequivalency of 2 gondatropin-releasing hormone (GnRH) preparations, gonadorelin diacetate tetrahydrate and gonadorelin semicarbonate, was compared on the basis of luteinizing hormone (LH)-releasing ability of the 2 products in diestrous dairy cows. Twenty-four cycling, nonlactating Holstein cows were subjected to a double prostaglandin estrus synchronization treatment to simultaneously control stage of the estrous cycle and time factors as potential variables effecting LH responses to the treatments being studied. Circulating progesterone concentration was determined to verify stage of cycle at strategic times throughout the study. Twelve days after the second prostaglandin treatment, all cows were randomly assigned to 1 of 2 groups (n = 12). Each group of 12 cows received single doses (100 micrograms) of either GnRH preparation at the start of each test period in a 2-period crossover design. Serum samples were obtained prior to and at 12 times (10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 480, and 1,440 minutes) after treatment and were assayed to determine circulating LH concentration. Significant difference between the 2 GnRH products was not found with respect to: mean concentration of LH in the blood during the 24 hours after treatment; maximal LH concentration; time from treatment to maximal LH concentration; and area under the LH concentration curve from time 0 through each of 7 times after treatment (0.5, 1, 1.5, 2, 4, 8, and 24 hours). These data confirm the bioequivalency of the 2 GnRH products.
Animals↗
Characterization of Mn(III) complexes of linear and cyclic desferrioxamines as mimics of superoxide dismutase activity.
The green complex of Mn(III) with desferrioxamine B has been prepared by air oxidation of Mn(II) in the presence of the ligand or by reaction of Mn(OH)3, or of MnO2, with the ligand. The resultant complex was purified by ion-exchange chromatography and shown by electrospray ionization mass spectrometry to be a 1:1 complex of Mn(III) with desferrioxamine B. A similar complex was prepared from the macrocyclic Tris hydroxamate desferrioxamine E. Mn(III) desferrioxamine B was not stable to equimolar EDTA while the Mn(III) desferrioxamine E was stable to a fivefold molar excess of EDTA. Chelex-100 decomposed the desferrioxamine B complex, but not the desferrioxamine E complex. These complexes prevented the reduction of cytochrome c by O2- but were less active than free Mn(II). The second-order rate constants for reaction with O2- at 25 degrees C and at pH 7.5 were 3.0 x 10(6), 1.0 x 10(6), and 9.5 x 10(4) for Mn(II), Mn(III) desferrioxamine B, and Mn(III) desferrioxamine E, respectively.
Catalysis↗
Application of electrospray ionization mass spectrometry for analysis of haemoglobin adducts with acrylonitrile.
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Acrylonitrile↗
The acceptor substrate specificity of porcine submaxillary UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is dependent on the amino acid sequences adjacent to serine and threonine residues.
The acceptor substrate specificity of a pure polypeptide N-acetylgalactosaminyltransferase has been examined with synthetic polypeptides with sequences identical, or similar to those found in porcine mucin or human erythropoietin. The sequences adjacent to either threonine or serine markedly influence the formation of GalNAc-O-Thr and GalNAc-O-Ser. Examination of the mucin-like peptide VLGXXAV, where X is Thr, Ser, or Ala, shows only Thr-containing peptides to be acceptors. The best substrate is formed when XX is TT. Peptides with XX as either AT or TA are less effective and those with XX as either ST or TS are much less effective acceptors. The amino acids adjacent to serine in the peptide formed by residues 121-131 in human erythropoietin, PPDAASAAPLR, also markedly influence the formation of GalNAc-O-Ser. Thus, PPDASSSAPLR and PPDVVSVVPLR are about 5- and 30-fold, respectively, less active than the erythropoietin peptide. The peptide PPDGGSGGPLR is inactive. The shorter peptide DAASAAPL is also about 5-fold less active than the full-length peptide, but the peptide AASAA is inactive. These studies indicate that one transferase can form both GalNAc-O-Ser and GalNAc-O-Thr residues when the sequences adjacent to the glycosylated residue are of the proper kind. Thus, in contrast to earlier suggestions, there is no evidence that different transferases form GalNAc-O-Ser and GalNAc-O-Thr. Examination of tissue homogenates from various tissues confirms this conclusion.
Amino Acid Sequence↗
Simultaneous injection of PGF2alpha and GnRH into diestrous dairy cows delays return to estrus.
Simultaneous injections of prostaglandin F2alpha (PGF) and gonadotropin releasing hormone (GnRH) or saline were given to 32 diestrous dairy cows to test the ability of GnRH to improve estrous and ovulation synchrony beyond that of PGF alone. Cows were randomly assigned to receive PGF on Day 8 or Day 10 of the estrous cycle (estrus = Day 0), and all cows were further assigned to simultaneous injection of GnRH or saline. Corpus luteum (CL) regression, return to estrus and follicular activity were monitored by plasma progesterone assay, twice-daily estrous detection and ultrasonographic examination, respectively. Plasma progesterone concentrations declined to <1.0 ng/ml at 24 hours after PGF in all cows and were not affected by GnRH. Gonadotropin releasing hormone inducted premature ovulation or delayed return to estrus in 7 of 8 cows treated with PGF/GnRH on Day 8 and 3 of 8 cows treated with PGF/GnRH on Day 10. Further, cows with premature GnRH-induced ovulations failed to develop and maintain a fully functional CL, and all returned to estrus 7 to 13 days after the induced ovulation. These data indicate that GnRH administered simultaneously with a luteolytic dose of PGF disrupts follicular dynamics and induces premature ovulation or delays normal return to estrus and, therefore, does not improve the synchrony of estrus and ovulation achieved with PGF alone.
Journal Article↗
Simultaneous injection of follicle stimulating hormone (FSH) and the prostaglandin F2alpha analog cloprostenol (PGF) disrupts follicular activity in diestrous dairy cows.
Simultaneous injections of PGF and FSH or saline were given to 32 Holstein cows to test their combined ability to improve estrous and ovulation synchrony beyond that of PGF alone. All the cows were randomly assigned to receive PGF on either Day 8 or Day 10 of the estrous cycle (estrus = Day 0), and all the cows in each group were further assigned to simultaneous injection of either FSH or saline. Regression of the corpus luteum (CL), return to estrus and follicular activity were monitored by plasma progesterone assay, twice-daily estrous detection and ultrasonographic examination, respectively. Plasma progesterone concentrations declined to <1.0 ng/ml at 24 hours after PGF treatment in all the cows and FSH did not affect this decline. Return to estrus was not affected by FSH treatment in cows treated on Day 8 or Day 10; however, FSH disrupted normal follicular activity and either delayed normal ovulation following estrus or induced premature ovulation or cyst formation in 4 of 8 PGF/FSH (Day 8) cows and 5 of 8 PGF/FSH (Day 10) cows. These data indicate that exogenous FSH administered simultaneously with a luteolytic does of PGF does not maintain viability of large, dominant follicles and, therefore, is not an effective method for the synchronization of estrus and ovulation.
Journal Article↗
Escherichia coli produces linoleic acid during late stationary phase.
Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.
Aerobiosis↗
Proton magnetic resonance studies of the aggregation of taurine-conjugated bile salts.
The concentration dependence of the 500 MHz 1H-NMR spectra of taurocholate, taurochenodeoxycholate, taurodeoxycholate, and the monosulfate esters of taurochenodeoxycholate has been examined at 0.154 M NaCl in D2O. The resonances of the C18, C19, and C21 methyl groups and the C23 methylene group are differentially broadened with respect to the C25 and C26 methylene and C7 (or C12) methine groups with increasing bile salt concentration for each of the bile salts studied. These data confirm hydrophobic association and indicate that the side chain contributes to the hydrophobic surface of the bile salt. The chemical shift difference of the anisochronous C23 methylene protons is different in monomer and aggregate form. The C25 methylene protons are isochronous in monomeric form but anisochronous in aggregate form. The concentration dependence of the observed chemical shifts has been analyzed to estimate the critical concentration associated with the onset of these changes. The conformer population about the C22-C23 bond changes before the anisochronicity of the C25 methylene protons develops. This indicates that the C23 methylene group is affected by the initial stages of self-association, whereas specific motional constraints about the N-C25 bond in the taurine moiety are only induced in large primary micelles. The difference in the chemical shift of the C25 methylene protons depends on the structure of the bile salt. The relative magnitude of the shift differences is not altered by the presence of phosphatidylcholine. The data suggest that in primary micelles or mixed micelles the taurine moiety conforms to segregate the hydrophilic groups of the bile salt and effects greater van der Waals' contact between the hydrophobic surfaces.
Bile Acids and Salts↗
Calcium binding by monosulfate esters of taurochenodeoxycholate.
The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.
Bile Acids and Salts↗
Effects of monosulfate esters of taurochenodeoxycholate on bile flow and biliary lipids in hamsters.
The effect of the 3 alpha- and 7 alpha-monosulfate esters of taurochenodeoxycholate on bile flow and biliary lipids was compared to the effect of unsulfated taurochenodeoxycholate. Test bile salts were infused directly into the portal circulation through a catheter introduced into the splenic pulp. Recovery of unsulfated and sulfated bile salts was complete; no biotransformation of any of the administered compounds was noted. Equivalent choleresis was noted in response to administration of each of the test bile salts. Of particular interest, the biliary cholesterol and phospholipid content was tightly linked to biliary bile salt monosulfates; the slope of the line describing the relationship between bile salts and lipids was similar to that for the unsulfated bile salt. The critical micellar concentration of the 3 alpha- and 7 alpha-monosulfate esters was 19 mM and 18 mM, respectively. Sulfation of taurochenodeoxycholate, therefore, does not impair its bile secretory function. Despite a higher critical micellar concentration, biliary lipid excretion with monosulfate esters is equivalent to that seen with unsulfated bile salt. The role of hydrophobic/hydrophilic balance in the promotion of biliary lipid excretion may need to be redefined.
Animals↗