Neural networks in enzyme engineering.
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Enzymes↗
Biomedical subjects
P Linko
Publications and source records attributed to P Linko.
A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability.
Cytochrome P450g is polymorphic in the male rat. This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes. Sequencing of cDNAs from high (+g) and low (-g) phenotype rats has shown that the low phenotype is due to a defective mRNA containing nine base mutations encoding 7 amino acid substitutions. To determine the role of these structural changes in the phenotypic expression of P450g, we altered each of these residues by site-directed mutagenesis in the present studies and expressed the normal and mutant cDNAs in Saccharomyces cerevisiae. P450+g protein was expressed at a level 4-6-fold higher than that of P450-g in yeast cells, despite the presence of identical mRNA levels. This difference in protein expression approaches the difference seen in the rat. A single amino acid change from Ser180 in P450+g to Cys in P450-g, in a highly conserved region in the P4502C subfamily, was found to be solely responsible for the phenotypic differences in expression of P450g. Protein half-life studies demonstrated that this mutation increases the degradation of P450g. This is the first example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability.
Amino Acid Sequence↗
Object-oriented fuzzy expert system for on-line diagnosing and control of bioprocesses.
An object-oriented fuzzy expert system to support on-line control of an automated fermentation plant is described. The major elements of the system consist of a fuzzy inference engine, a database, a knowledge base, and an expression evaluater. The expression evaluater calculates specific rates for growth, and substrate and product formation at different physiological states during the cultivation from the measured data. The specific rates are then compared with the standard target rates stored in the database. If differences outside the set tolerances were observed, the inference engine analyses the reasons for the faults on the basis of the knowledge represented in the form of a knowledge network and fuzzy membership functions of the process variables. The fuzzy expert system was developed on the basis of a shell constructed by using the object oriented Smalltalk/V Mac programming environment, with Lac-tobacillus casei lactic acid fermentation as the example of process application.
Biotechnology↗
Fuzzy reasoning system for fault diagnosis of physiological activities in a cultivating process.
Aiming at development of a system which supports cultivating operations, a method to diagnose physiological activities in a cultivating process is presented, and a fuzzy expert system for diagnosing Lactobacillus casei cultivating process is implemented in this paper. This system can calculate specific rates of cell growth, substrate consumption, and product formation with measuring cell mass concentration, substrate concentration, and product concentration by using a turbidity sensor and HPLC. A database is implemented, where standard curves on specific rates representing characteristics of microorganisms are stored according to normalized substrate consumption. Comparing the calculated specific rates with standard values derived from the database, the system diagnoses physiological activities of the microorganisms. As a case study, a knowledge base for diagnosing lactic acid production process is implemented. The use of fault diagnosis on pH malfunctions by the expert system proves its reasonable performance.
Biotechnology↗
Disposition of 2,3,7,8-tetrabromodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat: biliary excretion and induction of cytochromes CYP1A1 and CYP1A2.
The biologic activity and pharmacokinetic properties of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) are similar to those of the chlorinated congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Metabolism of both compounds appears to be rate-limiting for excretion, which is primarily via the feces. Therefore, the biliary elimination of TBDD and TCDD was examined as an indirect assessment of metabolism. Male F344 rats were anesthetized with pentobarbital, and 1 nmol/kg [3H]TBDD or [3H]TCDD was administered iv. Bile was collected for up to 8 hr while rats were maintained under anesthesia. The rate of biliary excretion of radioactivity was slightly greater for TCDD than TBDD (10% vs 7% in 5 hr). All biliary radioactivity was attributable to metabolites. High pressure liquid chromatographic (HPLC) profiles of biliary radioactivity were similar for [3H]TBDD and [3H]TCDD. To determine if pretreatment altered elimination kinetics, a single dose of 100 nmol/kg TBDD or TCDD was administered to rats by oral gavage 3 days prior to iv injection of 1 nmol/kg [3H]TBDD or [3H]TCDD, respectively. Biliary excretion of the radiolabeled dose was quantitatively and qualitatively unaffected by pretreatment despite a twofold increase in hepatic levels of radiolabel in the pretreated animals. Therefore, under these experimental conditions, autoinduction of TCDD and TBDD metabolism did not occur in the rat in vivo at doses which elicited enhanced hepatic uptake. In a second set of studies, the dose-response profiles for induction of cytochromes CYP1A1 and CYP1A2 by TBDD were characterized. The ED50 value for CYP1A1 induction (measured by ethoxyresorufin O-deethylase activity and radioimmunoassay (RIA) was estimated to be 0.8-1.0 nmol/kg, similar to what has been reported for TCDD. Induction of CYP1A2 (RIA) by TBDD appeared to be a more sensitive response over the dose range studied. Finally, comparison of hepatic CYP1A2 induction vs hepatic concentrations of TBDD 3 days following treatment with 10 vs 1 nmol/kg TBDD suggested that induction of CYP1A2 alone may not account for nonlinearities in dioxin disposition exemplified by dose-related increases in the ratio of dioxin concentrations in liver and adipose tissue.
Animals↗
Neural network programming in bioprocess variable estimation and state prediction.
A neural network program with efficient learning ability for bioprocess variable estimation and state prediction was developed. A 3 layer, feed-forward neural network architecture was used, and the program was written in Quick C ver 2.5 for an IBM compatible computer with a 80486/33 MHz processor. A back propagation training algorithm was used based on learning by pattern and momentum in a combination as used to adjust the connection of weights of the neurons in adjacent layers. The delta rule was applied in a gradient descent search technique to minimize a cost function equal to the mean square difference between the target and the network output. A non-linear, sigmoidal logistic transfer function was used in squashing the weighted sum of the inputs of each neuron to a limited range output. A good neural network prediction model was obtained by training with a sequence of past time course data of a typical bioprocess. The well trained neural network estimated accurately and rapidly the state variables with or without noise even under varying process dynamics.
Algorithms↗
Glutathione S-transferase mu in human lymphocyte and liver: role in modulating formation of carcinogen-derived DNA adducts.
Glutathione transferase (GT) activity towards trans-stilbene oxide (tSBO), benzo[a]pyrene-4,5-oxide (B[a]PO) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in human liver and lymphocytes. GT-tSBO activity is catalyzed by GT mu which has polymorphic expression in human lymphocytes. Our results show that activity of GT-tSBO in lymphocytes correlates with its activity in liver (r = 0.7, P less than 0.001). GT activity towards BPO (GT-BPO) also correlated with GT-tSBO in lymphocytes and liver. However, interindividual variation of GT-BPO is less than that of GT-tSBO, suggesting that BPO may not be as specific a substrate for GT mu and therefore other GT isozymes may contribute to BPO conjugation. Conjugation of CDNB by GT was not different using cytosols from either high or low GT mu individuals. The functional significance of the GT-mu polymorphism was evaluated by measuring its effect on benzo[a]pyrene (B[a]P)- and aflatoxin B1 (AFB1)-DNA adduct formation in vitro. Human liver cytosols prepared from persons having low or high GT-tSBO activity were incubated with human liver microsomes, calf thymus DNA and B[a]P or AFB1. HPLC analysis revealed that the major B[a]P adduct was dG(N2)-7 beta, 8 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). BPDE-dG adducts were decreased equally by cytosols from either low or high conjugators. In contrast, AFB1-DNA binding was inhibited to a greater extent in high conjugators than low conjugators. HPLC analysis demonstrates that adducts formed were AFB1-FAPyr and AFB1-N7-Gua. The correlation between AFB1-DNA adduct concentrations and GT mu activity was highly significant with a correlation coefficient of r = 0.88 at P less than 0.001. These results suggest that GT mu plays an important role in detoxifying DNA reactive metabolites of AFB1 and this enzyme may be a susceptibility marker for AFB1 related liver cancer. Moreover, our data demonstrate that lymphocytes are a reliable surrogate tissue for detecting liver GT mu polymorphisms.
Adolescent↗
Hormonal regulation of male-specific rat hepatic cytochrome P-450g (P-450IIC13) by androgens and the pituitary.
The present study examines the hormonal regulation male-specific cytochrome P-450g (IIC13) and its mRNA. Neonatal gonadectomy of male rats abolished hepatic expression of P-450g and its mRNA in adulthood, while ovariectomy had little effect. Neonatal administration of testosterone to neonatally gonadectomized male or female rats partially masculinized expression of P-450g and its mRNA, and postpubertal administration of testosterone (testosterone capsules implanted at 5 weeks) completely masculinized their expression. However, castration of male rats at puberty (5 weeks) had no effect on P-450g or its mRNA at 10 weeks. Male-specific development of P-450g and P-450 M-1 (IIC11) mRNA were imprinted similarly by testosterone. However, hypophysectomy experiments demonstrated that the two male-specific forms of P-450 are regulated quite differently. Hypophysectomy of male rats decreased hepatic content of P-450 M-1 mRNA by approximately 50%, and intermittent injections of growth hormone completely restored this mRNA. In contrast, hypophysectomy of male rats increased P-450g and its mRNA by approximately 50%, while intermittent injections of growth hormone produced a slight decrease. Hypophysectomy of female rats increased P-450g and its mRNA to adult male levels, but produced only a small increase in P-450 M-1 mRNA. Continuous infusion of growth hormone into sham hypophysectomized male rats (to mimic the female growth hormone pattern) resulted in a complete loss of P-450g and its mRNA. These results indicate that the expression of P-450g is not dependent on the male pulsatile growth hormone pattern, but suggest instead that the continuous secretion of growth hormone suppresses P-450g in the female rat.
Animals↗
Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist.
Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.
Animals↗
Suppression of male-specific cytochrome P450 2c and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl in rat liver is not causally related to changes in serum testosterone.
Rat cytochrome P450 2c (P450 gene IIC11) is a constitutive, male-specific hepatic enzyme which is suppressed greater than 90% by treatment with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) [H. N. Yeowell et al. (1987) Mol. Pharmacol. 32, 340-347]. HCB also decreases serum testosterone levels in adult male rats (greater than 98% loss). The present study assesses whether the suppression of P450 2c by HCB is a direct result of its effects on serum testosterone levels. Further, the site along the hypothalamic-pituitary-testicular axis at which HCB acts to depress testosterone secretion was examined. Administration of the synthetic androgen methyltrienolone to HCB-treated rats failed to prevent the suppression of P450 2c mRNA and its associated microsomal steroid 16 alpha-hydroxylase activity under conditions where it effectively reversed the large decrease in P450 2c mRNA and steroid 16 alpha-hydroxylase activity produced by castration. Hepatic steroid 6 beta-hydroxylase activity, which is catalyzed primarily by P450 2a (P450 gene IIIA2), was also suppressed by HCB and was not protected by methyltrienolone. Administration of either human chorionic gonadotropin, an analog of pituitary-derived luteinizing hormone, or the hypothalamic luteinizing hormone releasing hormone elevated serum testosterone levels to a much smaller extent in HCB-treated rats than in control rats. These results indicate that the effects of HCB on serum testosterone levels reflect its effects on testicular function rather than the pituitary or hypothalamus. However, the present study demonstrates that the consequential reduction in serum testosterone levels in HCB-treated rats is not causally related to the reduction in hepatic P450 2c levels. Thus, HCB must also act on some other regulatory mechanism involved in the expression of this protein.
Animals↗
The role of the Ah locus in hexachlorobenzene-induced porphyria. Studies in congenic C57BL/6J mice.
The role of the Ah locus in hexachlorobenzene (HCB)-induced porphyria and the possible involvement of P-450 cytochromes P(1)450 and P(3)450 in the pathogenesis of this disease were investigated in two congenic strains of C57BL/6J mice that differ only at this locus. Female B6-Ahb mice (Ah receptor: approximately 30-70 fmol/mg of cytosolic protein) and B6-Ahd mice (Ah receptor: undetectable) were pretreated with iron (500 mg/kg) and then fed a diet containing 0 or 200 p.p.m. of HCB for up to 17 weeks. Mice from the two strains consumed similar amounts of HCB. Urinary excretion of porphyrins was increased after 7 weeks of HCB treatment in B6-Ahb mice, and after 15 weeks was over 200 times greater than that of mice given iron only. In B6-Ahd mice, porphyrin excretion did not begin to increase until after 13 weeks, and after 15 weeks was only six times greater than that of controls. Similar differences were seen in the 15-week hepatic porphyrin concentrations (B6-Ahb: 1110 +/- 393; B6-Ahd: 17.6 +/- 14.5; controls: approximately 0.20 nmol/g). Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was diminished by 70 and 20% in B6-Ahb B6-Ahd mice respectively after 15 weeks of treatment with HCB. Cytochromes P(1)450 and P(3)450 were measured in hepatic microsomes (microsomal fractions) by radioimmunoassay and immunoblotting, using antisera raised against the orthologous rat isoenzymes P450c and P450d. HCB induced small amounts of a protein recognized by anti-P450c (P(1)450) in B6-Ahd mice, but not in B6-Ahd mice. Relatively large amounts of a protein recognized by anti-P450d (P(3)450) were induced in both strains, but to a somewhat greater extent in the B6-Ahb mice. The hepatic accumulation of HCB at 15 weeks was greater in B6-Ahb than in B6-Ahd mice, in association with elevated hepatic lipid levels in the former strain. The results of this experiment indicate that the Ah locus influences the susceptibility of C57BL/6J mice to HCB-induced porphyria and are consistent with the suggestion that the sustained induction of P(3)450 and/or P(1)450 may be a causative factor in the development of this disease.
Animals↗
Increases in cytochrome P-450 mediated 17 beta-estradiol 2-hydroxylase activity in rat liver microsomes after both acute administration and subchronic administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a two-stage hepatocarcinogenesis model.
Administration of single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10 micrograms/kg) increased estradiol 2-hydroxylase (E2OHase) activity approximately 2-fold in liver microsomes of female rats but had no effect on E2OHase activity in hepatic microsomes of male rats. In contrast, TCDD increased P-450d (an enzyme which has a high turnover number for E2OHase in a reconstituted enzyme system) 10- to 20-fold in livers of both male and female rats. The discrepancy between the increases in P-450d and E2OHase activity in liver microsomes of TCDD-induced rats was abolished by the addition of exogenous purified P-450 reductase to the microsomal assays for E2OHase, suggesting that reductase was limiting in the in vitro assays. When E2OHase activity was assayed in the presence of exogenous reductase, TCDD increased E2OHase 2-fold and 4-fold respectively in liver microsomes of male and female rates. Antibody to P-450d completely inhibited the increase in E2OHase activity in liver microsomes of TCDD-treated male and female rats, but had little effect on E2OHase activity in liver microsomes of untreated male or female rats. These data indicate that P-450d is responsible for the increase in E2OHase activity in TCDD-treated rats, but other P-450 isozymes are responsible for constitutive E2OHase activity. Biweekly administration of 1.4 micrograms/kg of TCDD for 30 weeks as a potential promoter of hepatocarcinogenesis increased the volume of the liver occupied by gamma-glutamyl transpeptidase (GGT)-positive foci in livers of female rats given a necrogenic dose of diethylnitrosamine (DEN) (200 mg/kg) as the initiator. Biweekly doses of 0.14-1.4 micrograms/kg TCDD in this model also increased P-450d (7-fold) and E2OHase activity maximally (4-fold) in DEN-initiated rats. Moreover, initiation with DEN substantially enhanced the effects of the low dose of TCDD on both hepatic microsomal P-450d and E2OHase activity.
Animals↗
Uncertainties, fuzzy reasoning, and expert systems in bioengineering.
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Biomedical Engineering↗
Induction of rat cytochrome P-450 3 and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl.
Rat cytochrome P-450 3 (P-450 3) is a constitutive hepatic steroid hormone 7 alpha-hydroxylase which is relatively unresponsive to a number of monooxygenase-inducing agents. The present study demonstrates that a polyhalogenated aromatic hydrocarbon inducer, 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), induces P-450 3 in livers of adult male rats, and that the increase is the result of an increase in the mRNA for this enzyme. Cytochrome P-450 3 and its mRNA were increased more slowly and to a lesser extent than cytochrome P-450c (P-450c) and its mRNA, indicating that these enzymes are not regulated coordinately in liver. The maximum increase in P-450 3 and P-450 3-dependent androstenedione 7 alpha-hydroxylase activity (2- to 3-fold) occurred 7 days after administration of HCB, in contrast to the increase in P-450c (greater than 200-fold) which was maximal by 3-5 days. The rate of induction of P-450 3 mRNA was also slower [maximum increase (9-fold) at 5 days after HCB administration] than that of P-450c mRNA [maximum increase (30-fold) at 2-3 days]. Moreover, a higher dose of HCB was required to produce maximum induction of P-450 3 (50 mg/kg) than that required to produce maximum induction of P-450c (10 mg/kg). P-450 3 was not detected on Western blots of lung, kidney, or prostate microsomes isolated from control or HCB-treated rats (less than or equal to 2% of that found in livers of HCB-treated rats). Moreover, P-450 3-dependent steroid 7 alpha-hydroxylase activity was not detected in these extrahepatic tissues of control or HCB-treated rats (less than or equal to 1% of that found in the corresponding liver microsomes of untreated or HCB-treated rats). In contrast, P-450c was increased dramatically by HCB in lung, kidney, and prostate tissues, indicating differential expression of P-450c and P-450 3 in extrahepatic tissues.
Animals↗
Entrapment of microbial cells in cellulose gel.
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Aldose-Ketose Isomerases↗
Induction of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah)-responsive mice.
Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At greater than 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70 chi), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58 chi) and P-450d (8 chi), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ah-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P3-450 and a very small increase in P1-450 in the responsive strain. The increase in P1-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P3-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [3H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [3H]-TCDD only at very high concentrations (10(-6) to 10(-5) M).
Animals↗
2,3,7,8-Tetrachlorodibenzofuran in a commercially available 99% pure polychlorinated biphenyl isomer identified as the inducer of hepatic cytochrome P-448 and aryl hydrocarbon hydroxylase in the rat.
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Animals↗