Biomedical subjects
Publications and source records attributed to G S Bailey.
Kininase (kinin-degrading), tyrosine esterase, kininogenase (kinin-releasing) and arginine esterase activities of various crotalid, viperid and elapid venoms were measured. Wide ranges of those enzymatic activities were recorded for the crotalid and viperid venoms but no activities were detected in the Naja venoms.
The effectiveness of ammonia in inactivating aflatoxins in contaminated cottonseed was investigated. Two aflatoxin-contaminated cottonseed lots were treated separately using an atmospheric pressure, ambient temperature ammoniation procedure (APAT) or a high pressure, high temperature ammoniation procedure (HPHT), and incorporated into dairy cow rations. Isocalorific diets containing 25% defatted, dried milk from cows fed aflatoxin-contaminated cottonseed without or with APAT or HPHT treatment, or an aflatoxin-free human grade commercial milk powder, were then fed for 12 months to rainbow trout (Oncorhynchus mykiss). Aflatoxin M1 (AFM1) concentrations in milk powders without and with seed treatment were: APAT, 85 and < 0.05 microgram/kg; HPHT, 32 and < 0.05 microgram/kg. In the APAT experiment, trout consuming the diet containing milk from cows fed the aflatoxin-contaminated cottonseed had a 42% incidence of hepatic tumours; APAT cottonseed treatment reduced this to 2.5%. Positive controls were included to demonstrate trout responsiveness. AFB1 fed continuously for 12 months at 4 micrograms/kg resulted in a 34% tumour incidence, whereas positive controls fed 20 micrograms AFB1/kg, 80 micrograms AFM1/kg, or 800 micrograms AFM1/kg for 2 wk and killed 9 months later had a 37, 5.7 and 50% incidence of tumours, respectively. These data demonstrate that APAT ammonia treatment of aflatoxin-contaminated dairy cattle cottonseed feedstock abolished the detectable transfer of AFM1 or AFB1 into milk powder, and greatly reduced the carcinogenic risk posed by any carry-over of aflatoxins or their derivatives into milk. In addition, the results confirm AFM1 to be a lower level hepatocarcinogen in comparison with AFB1 in the trout carcinogenicity assay. In the separate HPHT experiment, no tumours were observed in the livers of trout fed diets containing milk from either the ammonia-treated or untreated source, or the control diet containing 8 micrograms AFM1/kg. Positive controls fed 64 micrograms AFB1/kg for 2 wk exhibited a 29% tumour incidence 12 months later. Thus in this experiment, neither AFM1 at 8 micrograms/kg nor any HPHT-derived aflatoxin derivatives that might have been carried over into milk, represented a detectably carcinogenic hazard to trout.
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1. A hydrophilic and a hydrophobic prokallikrein were purified from sheep kidney cortex and some of their biochemical properties were compared. 2. The effects of several inhibitors on the activity of the hydrophilic and hydrophobic kallikreins were investigated. 3. The thermal stability and response of the hydrophobic kallikrein towards various detergents were also studied.
Two kallikreins were identified in a homogenate of bovine pancreas in terms of their differential elution from an anion-exchange chromatography column. The kallikreins were quantified by their ability to release kinin from a partially purified preparation of bovine kininogen. The second kallikrein, designated kallikrein B, was purified by a three-step procedure following anion-exchange chromatography consisting of affinity chromatography on a benzamidine-agarose resin, gel filtration and hydrophobic interaction chromatography. An overall purification factor of 556-fold was achieved with a 58% recovery of enzymatic activity. The final material was shown to be homogeneous by a number of electrophoretic analyses. The relative molecular mass of pro-kallikrein B was found to be 26,700 by gel filtration and that of kallikrein B to be 26,000 by SDS gel electrophoresis. Gel isoelectric focusing revealed the presence of several isoenzymic forms ranging in isoelectric point from pH 4.05 to 4.35.
Field studies suggest that recent epizootics of hepatic neoplasms in some feral fish populations are associated with polycyclic aromatic hydrocarbon (PAH) exposure, but attempts to induce liver tumors in these species under laboratory conditions have been unsuccessful. Several studies have shown hepatic neoplasma to be inducible in laboratory fish species following PAH exposure at the free-swimming life stage. However, neither the susceptibility of the fish embryonic life stage to tumor induction by PAHs nor the potential of these carcinogens to induce oncogenic point mutations analogous to those reported in feral fish hepatic tumors have been clearly established. To address this, rainbow trout embryos were exposed by passive water uptake to 7,12-dimethylbenz[a]anthracene (DMBA), a potent model PAH in many mammalian tumor protocols. DMBA was rapidly absorbed by trout eggs and metabolized. The major non-polar metabolites identified were 12-hydroxymethyl-7-methylbenz[a]anthracene and 3,4-dihydroxy-3,4-dihydro-DMBA, whereas approximately 25% of the water soluble metabolites were identified as glucuronides by beta-glucuronidase treatment. Embryonic DNA adduction increased with time of DMBA exposure (2.2 +/- 0.3 pmol DMBA-equivalents/mg DNA at 24 h). Liver tumor incidence nine months after DMBA treatment was found to increase with DMBA concentration and exposure period (3.8% at 1 p.p.m./2 h; 23% at 5 p.p.m./2 h; 85% at 5 p.p.m./24 h). Stomach adenomas and nephroblastomas also were observed at low incidence in the DMBA-treated trout. Among 11 hepatic tumors examined, nine carried Ki-ras alleles with activating point mutations in codon 12 (4/11 GGA-->AGA; 4/11 GGA-->GTA) or codon 61 (1/11 CAG-->CTG). This spectrum differs substantially from those reported for DMBA-initiated mouse skin papillomas or hepatic tumors. These results may have important environmental implications because they suggest that even a brief exposure to PAHs during a sensitive stage of development may adversely affect some fish populations. They also indicate considerable variation in DMBA ras gene mutations among species and target organs.
Tonin was isolated from rat submandibular glands by a very convenient procedure consisting of sequential anion-exchange, hydrophobic interaction and gel filtration chromatographies. The method is superior to earlier purifications as it consists of fewer stages, resulting in a much higher recovery (41%) of tonin. The final preparation was seen to be pure on SDS gel electrophoresis (M(r) 32,800) and on gel isoelectric focusing (pI 6.15). The stability of tonin and its interaction with various inhibitors were investigated, and compared with the corresponding behavior of rat tissue kallikrein.
The 1H-NMR spectra have been obtained for rat submandibular kallikrein in the absence and presence of inhibitors. Two competitive inhibitors were investigated, the tripeptide leupeptin (a potent inhibitor with Ki 0.5 microM) and a hexapeptide (a much weaker, substrate-analogue inhibitor with Ki 380 microM). Analysis of the NMR spectra showed that binding of leupeptin to kallikrein led to a change in the conformation of the enzyme, whereas binding of the substrate analogue to the enzyme produced no such change and may have resulted in a conformational change of the inhibitor.
Numerous biochemical properties (e.g. Mr, carbohydrate content, pI) were determined for kallikrein isolated from rat submandibular glands by a simple, rapid purification procedure. The kinetic behaviour of the enzyme towards various inhibitors and synthetic substrates was investigated. The effects of different salts and detergents on the esterolytic activity of the rat tissue kallikrein were recorded.
Kallikrein was isolated from bovine pancreatic tissue by a sequential procedure of anion-exchange, hydrophobic interaction and gel filtration chromatographies. A monospecific polyclonal antiserum to the pure enzyme was raised in rabbits and was used to set up a specific radioimmunoassay for bovine tissue kallikrein.
We have used assays for histamine and for the specific mast cell enzyme, tryptase, to examine the response of the nasal mucosa to provocation with several different stimuli and to evaluate the reliability of histamine as a marker of mast cell activation. High levels of histamine detected in baseline lavages of some subjects are not associated with any detectable tryptase, suggesting they are not mast cell derived. During pronounced immediate allergic responses, however, mast cell degranulation clearly occurs, and a striking correlation between histamine and tryptase is observed. This correlation is weaker when a more modest allergic response is induced, possibly reflecting differential diffusion of the two mediators across the epithelium. Provocation of susceptible individuals with cold, dry air leads to increased recoveries of both histamine and tryptase, confirming that mast cell degranulation occurs during this reaction. Although hyperosmolarity of the nasal mucosa may contribute to mast cell degranulation induced by cold, dry air, a brief exposure of the nasal cavity to hyperosmolar mannitol was not associated with measurable production of tryptase. The combined use of histamine and tryptase measurements can therefore provide useful evidence regarding the role of mast cell activation in the pathogenesis of inflammatory responses.
A prokallikrein was isolated from bovine pancreas by a multi-step procedure involving gel filtration, hydrophobic interaction and anion-exchange chromatographies. The purification was initially monitored by measurement of the kinin-releasing activity of the activated zymogen. Later, when the pure prokallikrein had been isolated, a specific radioimmunoassay for the zymogen was set up and that was employed to provide estimates of 323-fold and 28% for the overall degree of purification and percentage recovery of prokallikrein. The relative molecular weight of prokallikrein was found to be 26,900 by SDS gel electrophoresis and its isoelectric point was established as pH 4.55.
Indole-3-carbinol (13C), a secondary metabolite from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in trout (Bailey et al., J. Natl. Cancer Inst., 78: 931-934, 1987) and rats (Selivonchick et al., unpublished results) when given prior to and with carcinogen but promotes carcinogenesis in both species when given continuously following AFB1 initiation. Since human 13C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed 13C promotion using delayed and discontinuous exposure protocols. Following initiation with AFB1, 13C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermittent patterns of 13C treatment. Promotional enhancement of tumor incidence by 13C was found to be significant when 13C treatment was delayed for several weeks or months after the initial AFB1 challenge. Promotion also was found to increase with length of exposure to 13C treatment and to be decreased but still evident when 13C was given in alternating months or weeks, or twice per week only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify 13C promotional potency in terms of its dietary concentration, a series of AFB1 tumor dose-response curves was established, each with a different level of 13C fed continuously following AFB1 initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log AFB1 dose, were displaced parallel toward lower AFB1 50% tumor take (TD50) values with increasing 13C concentration. The level of 13C that halves the AFB1 dose for 50% tumor incidence was calculated to be approximately 1000 ppm 13C, fed continuously, with no substantial threshold for promotion. By comparison, 13, when fed before and with AFB1, shows a 50% inhibitory value (13C concentration that doubles the dose of AFB1 for 50% tumor incidence) in trout of 1400 ppm 13C [Dashwood et al., Carcinogenesis (Lond.), 10: 175-181, 1989]. Thus the potential for 13C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFB1 initiation.
The activities of kinin-releasing enzymes in the venoms of Vipera xanthina xanthina, V. lebetina obtusa, V. aspis aspis, V. lebtina schweizeri, V. ammodytes ammodytes and V. berus berus were determined using a specific radioimmunoassay for kinin. The kinin-releasing activities of all the viperid venoms measured in vitro were neutralized, to varying extents, by two commercially available monospecific antivenoms in the form of F(ab')2 (Zagreb) and Fab (TAb) immunoglobin fragments, indicating a high degree of cross-neutralization of those enzymes.
The amounts of kinin-releasing enzymes in the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus scutulatus and Agkistrodon piscivorus piscivorus were measured by determining the amounts of kinin released from a sheep kininogen substrate by means of a specific radioimmunoassay. Four monospecific and two commercial polyspecific antivenom IgG samples were tested for their ability to reduce the kinin-releasing activities of the four crotalid venoms measured in vitro. All of the antivenom IgG samples were able to neutralize venom kininogenase activity to varying extents. On of the commercial polyspecific antivenoms was of equal or higher potency than the corresponding monospecific antivenoms for three of the venoms tested, indicating a high degree of cross-neutralization. F(ab')2 and especially Fab fragments of that polyspecific antivenom IgG were also effective in reducing the kinin-releasing activities of the four crotalid venoms.
The pharmacokinetics, tissue distribution and excretion of 14C-labelled aflatoxin B1 (AFB1) were examined after oral administration (250 micrograms/kg body weight) in channel catfish (Ictalurus punctatus). Plasma concentrations of parent AFB1 were best described by a one-compartment pharmacokinetic model, in which peak plasma concentration (503 ppb) occurred at 4.1 hr after dosing. The absorption and elimination half-lives were 1.5 and 3.7 hr, respectively. AFB1 was highly bound (95%) to plasma proteins. Concentrations of 14C (in AFB1 equivalents) measured in the tissues were highest at 4 hr, ranging from 596 ppb in the plasma to 40 ppb in the muscle. AFB1 residues were rapidly depleted; at 24 hr the concentrations in the plasma and muscle were 32 and less than 5 ppb, respectively. Concentrations in the bile exceeded 2000 ppb (at 24 hr), whereas the highest concentration in the urine was 51 ppb (4-6-hr collection interval). Renal and biliary excretion accounted for less than 5% of the administered dose, indicating incomplete absorption. Pharmacokinetic modelling and tissue data demonstrate a very low potential for the accumulation of AFB1 and its metabolites in the edible flesh of channel catfish through the consumption of AFB1-contaminated feed.