Hepatic Poly Research Articles

Large Supplements of Nicotinic Acid and Nicotinamide Increase Tissue NAD+ and Poly(ADP-Ribose) Levels but Do Not Affect Diethylnitrosamine-Induced Altered Hepatic Foci in Fischer-344 Rats

Poly(ADP-ribose) is a homopolymer of ADP-ribose units synthesized from NAD+ on nuclear acceptor proteins and is known to be involved in DNA repair. It is not known whether large oral doses of the clinically utilized NAD precursors nicotinic acid or nicotinamide affect poly(ADP-ribose) metabolism or the cellular response to DNA damage. In our first study, using Fischer-344 rats, 2 wk of dietary nicotinic acid supplementation (500 and 1000 mg/kg diet) caused elevated levels of NAD+ in the blood, liver, heart and kidney, while nicotinamide caused elevated levels only in the blood and liver, compared with controls fed a diet containing 30 mg/kg nicotinic acid. Both nicotinic acid and nicotinamide, at 1000 mg/kg diet, caused elevations in liver NAD+, by 44 and 43%, respectively. Only nicotinamide, however, elevated liver poly(ADP-ribose) (63% higher than control group). Following treatment with the hepatocarcinogen diethylnitrosamine, higher levels of hepatic NAD+ were observed in rats fed both nicotinic acid and nicotinamide at 1000 mg/kg diet, but only nicotinic acid supplementation caused a greater accumulation of hepatic poly(ADP-ribose) (61% higher than control group). Neither of the dietary treatments significantly affected the proportion of the liver occupied by placental glutathione-S-transferase positive foci. These results show that poly(ADP-ribose) synthesis is not directly responsive to hepatic NAD+ levels during niacin supplementation, and that the mechanisms of action of nicotinic acid and nicotinamide are different. The observed changes in poly(ADP-ribose) metabolism do not appear to cause any change in susceptibility to chemically induced carcinogenesis in this organ.

Poly(ADP-Ribose) Polymerase Activity and DNA Strand Breaks Are Affected in Tissues of Niacin-Deficient Rats

The niacin cofactor, NAD, is the substrate for poly(ADP-ribose) polymerase, an enzyme associated with DNA repair. We investigated, therefore, whether hepatic poly(ADP-ribose) polymerase activity was altered and DNA strand breaks in lymphocytes and liver were greater in niacin-deficient rats. A niacin deficiency was established in weanling rats with diets containing 1.5 mg/kg of niacin. Based on lower growth rates and NAD concentrations in blood, liver and skeletal muscle, this diet maintained rats in a deficient state for 1 mo, and, when the dietary niacin was reduced to 0.5 mg/kg, rats remained deficient for an additional month. The hepatic poly(ADP-ribose) polymerase activity was decreased in one experiment when mean hepatic NAD concentrations were 0.60 and 0.51 mumol/g at d 34 and d 60, respectively, compared with 0.77 and 0.80 mumol/g in pair-fed controls. Enzyme activity, however, was greater than in controls when hepatic NAD concentrations were < 0.30 mumol/g. Strand breaks in DNA did not accumulate except after tissues were exposed to hypoxanthine-xanthine oxidase, a free radical-generating system. Exposure to this system caused more DNA strand breaks in lymphocytes and hepatic nuclei from niacin-deficient rats compared with the same tissues from controls. The results suggest that, in rats, although hepatic poly(ADP-ribose) polymerase activity can be elevated, a severe niacin deficiency may increase the susceptibility of DNA to oxidative damage, likely due to a lower availability of NAD.

Propolis ethanol extract abrogates hyperglycemia, lipotoxicity, and lowered hepatic poly (ADP-ribose) polymerase protein level in male albino rats.

Diabetes is a major cause of death worldwide and currently available allopathic drugs presents adverse side effects, thus, necessitating a continuous screening for natural products. This study therefore investigated the effects of Propolis Ethanol Extract (PEE) on blood sugar, lipid metabolism, and poly-(ADP)-ribose polymerase (PARPs) protein level of diabetic male Wistar rats. Seventy rats weighing between (150-180) g used in this study were randomized into seven (7) groups as follows: group 1 (Normal control given Olive oil), group 2 (Diabetic control given Olive oil), group 3 [Diabetic + PEE (200mg/kg)], group 4 [Diabetic + (PEE 600mg/kg)], group 5 [Diabetic + Glibenclamide (10mg/kg)], group 6 [Normal + PEE (200mg/kg)], and group 7 [Normal + PEE (600mg/kg)]. Diabetes was induced by a single intraperitoneal injection of streptozotocin (65mg/kg in 0.1M citrate buffer pH 4.5), while the vehicle and PEE were orally administered once daily. Treatment with PEE commenced after the confirmation of diabetes. Five rats from each group were sacrificed after the third and sixth weeks of PEE treatment. Administration of PEE significantly (P < 0.05) lowered the elevated fasting blood sugar, improves body weight, and abated lipotoxicity in the brain, heart, liver and kidney of the treated groups in a dose- and duration-dependent manners. The increased protein level of PARPs and lowered hydroxyl methyl-glutaryl CoA reductase activity were significantly reversed after PEE treatment. This study concludes that PEE might be a suitable and viable regimen against diabetic complications in rats.

Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

Low Inducibility of CYP1A Activity by Polychlorinated Biphenyls (PCBs) in Flounder (Platichthys flesus): Characterization of the Ah Receptor and the Role of CYP1A Inhibition

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolk AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (˜2–7 fmol/mg protein) were much lower than observed generally in rodents (˜50–300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59–60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducibk TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorod-ibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat The Km for ethoxyresonifin (0.095 μM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresonifin than that of rat CYP1A.

Nicotinamide Megadosing Increases Hepatic Poly(ADP-Ribose) Levels in Choline-Deficient Rats

Previous work in our laboratory has shown that dietary megadoses of nicotinamide, used in the prevention of diabetes, cause increases in hepatic poly(ADP-ribose). Poly(ADP-ribose) is synthesized from NAD+ by a nuclear enzyme, poly(ADP-ribose)poly-merase, which is activated by DNA strand breaks. The nicotinamide-induced increase in poly(ADP-ribose) could result from an increase in substrate, NAD+, or the induction of strand breaks in DNA. Strand breaks may result from the depletion of single carbon groups, through the excretion of methylated derivatives of nicotinamide. To differentiate between these mechanisms, a 3 ՠ3 factorial experiment was conducted in which rats were fed diets containing various supplements of choline bitartrate (0, 2, 20 g/kg diet) and nicotinamide (0, 1, 2 g/kg diet). At the conclusion of treatments, blood NAD+ and liver lipid, NAD+ and poly(ADP-ribose) levels were determined. Choline deficiency caused the characteristic accumulation of fat in the liver at all levels of nicotinamide. In choline deficient rats, nicotinamide supplements further increased liver lipid concentration. Blood and liver NAD+ concentrations were increased by nicotinamide supplementation, irrespective of choline status. In contrast, liver poly(ADP-ribose) levels were increased by nicotinamide supplementation only in choline deficient rats. These results show that nicotinamide-induced increases in poly(ADP-ribose) levels appear to be dependent on decreased methyl donor status and suggest that adequate choline status is important for preventing some deleterious effects of nicotinamide treatment.

The effect of niacin deficiency on diethylnitrosamine‐induced hepatic poly(ADP‐ribose) levels and altered hepatic foci in the fischer‐344 rat

Poly(ADP-ribose) is synthesized on nuclear proteins in response to DNA damage and plays an important role in DNA repair. Niacin and tryptophan are dietary precursors to NAD+, which is the substrate for poly(ADP-ribose) synthesis. This study examined the influence of niacin status on poly(ADP-ribose) metabolism and carcinogenesis. Diets devoid of added niacin, with different levels of tryptophan, were used to produce moderate and severe niacin deficiencies in male Fischer-344 rats. Control rats were pair fed niacin-replete diets. After a 21-day feeding period, rats were injected with diethylnitrosamine (DEN) (Expt 1, 200 mg/kg ip; Expt 2, 100 mg/kg ip). In Experiment 1, blood and liver NAD+ and liver poly(ADP-ribose) were measured over the next 15 hours. Whereas blood and liver NAD+ were decreased by niacin deficiency, blood NAD+ was not affected by DEN. Liver NAD+ decreased significantly in response to DEN treatment in the pair-fed groups, but it did not change in the niacin-deficient groups. Unexpectedly, at 10 hours postinjection, liver poly(ADP-ribose) accumulation was greater (p < 0.05) in the niacin-deficient than in the pair-fed rats (n = 9), despite lower initial NAD+ levels and a lack of NAD+ disappearance in niacin-deficient livers. In Experiment 2, livers were examined for the presence of altered hepatic foci three months after DEN exposure. There were no significant differences in the percentage of liver occupied by foci between the niacin-deficient and pair-fed groups (n = 8). These results indicate that niacin-deficient rats were able to accumulate higher concentrations of hepatic poly(ADP-ribose) in response to DEN and did not show elevated susceptibility to initiation of altered hepatic foci.

Hepatic Poly(ADP-Ribose) Polymerase Activity in Rat Is Controlled by Thyroid Hormones

The in vivo effect of the thyroidal state on poly(ADP-ribose) polymerase activity was investigated in eu- and hypothyroid rats after treatment with L-triiodothyronine. Untreated hypothyroids showed an increased basal rate of the enzyme. The treatment of both eu- and hypothyroid rats with L-triiodothyronine induced a prompt drop of the endogenous activity not due to a reduction of the catalytic protein. This decrease well evident 1 h after treatment was transient, returning to controls values within 8 h. In isolated liver nuclei from euthyroids the in vitro exposure to increasing L-triiodothyronine concentrations from 10−18 to 10−6 M resulted in a progressive inhibition of the enzyme. This loss in activity was not derived from a reduction of the total level of the catalytic protein. The pretreatment with the antagonist amiodarone suppressed the hormone effect,suggesting that nuclear receptors could mediate poly(ADP-ribose) polymerase activity.

Diethylnitrosamine administration in vivo increases hepatic poly(ADP-ribose) levels in rats: results of a modified technique for poly(ADP-ribose) measurement.

Poly(ADP-ribose) (polymer) is enzymatically synthesized on nuclear proteins in response to DNA strand breaks. NAD+ is the substrate for this reaction, which is catalyzed by poly(ADP-ribose)polymerase. This post-translational modification occurs in response to DNA strand breaks and is thought to play an important role in DNA repair. Polymer synthesis resulting from DNA damage has been described in cultured cells, but measurement is more difficult in animal tissues. In this study, modifications were made to an earlier method to measure carcinogen-induced increases in polymer levels in vivo. RNase I was added to the enzyme mixture used to digest polymer to ribosyladenosine (RAdo). This prevented the inhibition of snake venom phosphodiesterase by RNA. The HPLC analysis was improved, allowing elimination of the second boronate affinity chromatography step traditionally used to purify epsilon RAdo. Using this technique, we have studied the effect of i.p. diethylnitrosamine (DEN) injection on hepatic NAD+ and poly(ADP-ribose) levels in Fischer-344 rats. Hepatic polymer levels rose 8-fold from 26 to 218 pmol/g liver wet weight, 10 h following 200 mg DEN/kg body weight (n = 4-5). Liver NAD+ decreased concurrently, to 61% of basal levels at 16 h post-treatment (n = 4-5). Erythrocyte NAD+ concentrations remained unchanged, despite carcinogen administration. The DEN-induced effects on tissue polymer and NAD+ levels were dose dependent from 0 to 200 mg DEN/kg body weight (n = 4).

Gene expression of insulin-like growth factors (IGFs), the type 1 IGF receptor, and IGF-binding proteins in dexamethasone-induced fetal growth retardation.

Altered gene expression and/or actions of the insulin-like growth factors (IGFs) have been implicated in the mediation of both pre- and postnatal growth retardation secondary to glucocorticoid excess. To investigate this possibility, we assessed the gene expression of the IGFs, the type I IGF receptor, and IGF-binding proteins (IGFBPs) in 20-day gestation liver and lung of growth-retarded fetuses whose mothers were treated with dexamethasone (DXM; 100 micrograms, ip, daily) on gestation days 15-19 (gestation = 21-22 days). DXM treatment in dams produced fetal growth retardation without decreasing litter size (32% decrease in fetal body weight). Both fetal liver and lung demonstrated decreased wet weight (48% and 47%, respectively) and DNA content (65% and 51%, respectively) compared to control animals. Our results suggest that increased expression of IGFBP-1, and possibly IGFBP-2, is involved in mediating the marked growth retardation. As assessed by solution hybridization assays and Northern blot analysis, there was an 8.5-fold increase in IGFBP-1 mRNA expression in the livers of DXM-treated fetal animals compared to that in sham-injected controls (P less than 0.002). IGFBP-2 mRNA expression was also increased (60%) in fetal liver, whereas IGFBP-3 was decreased (57%). In fetal lung, IGFBP-1 transcript abundance was also higher in DXM-treated fetal animals. Serum concentrations of IGFBP-1, but not those of IGFBP-2, were increased (approximately 4-fold) in the DXM-treated fetuses, as quantified by [125I]IGF-I ligand blotting and IGFBP-2 immunoblotting. Because hypoinsulinemia increases the expression of IGFBP-1 and -2, serum insulin concentrations were measured and found to be decreased in the DXM-treated fetuses (24 microU/ml) compared to control values (72 microU/ml). Analysis of mRNA expression for IGF-I, IGF-II, and the type 1 receptor transcripts did not support a role for decreased IGF or IGF receptor expression in the etiology of DXM-mediated growth retardation. IGF-I was unchanged in both liver and lung, and IGF-II transcripts were increased by 31% in liver and unchanged in lung of DXM-treated fetal animals. Northern analysis of hepatic and lung poly(A) RNA demonstrated no evidence for independent regulation of specific-sized IGF transcripts. Further, type 1 IGF receptor RNA abundance increased 42% in fetal liver and was unchanged in lung. Because IGFBPs may modulate IGF action, these results suggest that increased IGFBP-1, and possibly IGFBP-2, expression may be of importance in the etiology of DXM-induced fetal growth retardation.

In vitro translational activity of messenger-RNA isolated from mice treated with the interferon inducer, polyriboinosinic acid·polyribocytidylic acid

Treatment of mice with interferon and interferon inducers causes down regulation of a number of hepatic proteins. In a previous publication it was demonstrated that these treatments depress hepatic protein synthesis and increase protein degradation, particularly of the endoplasmic reticulum Gooderham NJ and Mannering GJ, Arch Biochem Biophys 250: 418–425, 1986. In the present study the effects of polyriboinosinic acid-polyribocytidylic acid (poly IC) treatment on mouse hepatic RNA levels and the translation of this RNA in a cell-free system were examined. Poly IC treatment of mice increased hepatic poly (A +)RNA levels. The translation of isolated poly(A +)RNA was evaluated at various intervals after the administration of poly IC. Translation was marginally increased at 3–6 hr after treatment and depressed after 12–18 hr. Antibodies were employed to examine the effects of poly IC treatment on specific polypeptides in order to evaluate the in vitro translation of mRNAs for tyrosine aminotransferase and albumin; translation of these proteins was biphasic with pronounced depression. These studies indicate that in vivo interferon may regulate gene expression by altering levels of hepatic proteins via increased transcription and decreased translation.

In Vivo Regulation of Hepatic Insulin-Like Growth Factor-1 Messenger Ribonucleic Acids with Thyroid Hormone.

Insulin-like growth factor-1 (IGF-1) is synthesized primarily by the liver in response to growth hormone (GH). Thyroid hormone plays a major role in mediating pituitary GH secretion. In order to clarify the effect of thyroid hormone on IGF-1 gene expression, we measured hepatic IGF-1 mRNA levels in rats with thyroid dysfunction. Female Wistar rats were rendered hypothyroid by surgical thyroidectomy or hyperthyroid by daily injections of thyroxine (12 micrograms/day) for 2 weeks. Northern gel analysis of hepatic poly (A) RNA revealed the multiple sizes of the RNA transcripts ranging from 1.6 to 9.0 kb. After 4 weeks, hepatic IGF-1 mRNA levels were suppressed in hypothyroid rats, to less than 20% of control euthyroid animals. These suppressed mRNA levels were restored to euthyroid levels by thyroid hormone replacement for 2 weeks. Hyperthyroid rats, however, did not contain altered levels of hepatic IGF-1 mRNA as compared to euthyroid rats. The gamma-actin mRNA hybridization signal was not altered in hypothyroid or hyperthyroid rats. These results suggest that thyroid hormone regulates the in vivo expression of hepatic IGF-1 mRNA, probably through the mechanism of GH regulation.

Hepatic Poly(ADP ribose) Polymerase Activity in Methyl Donor—Deficient Rats

Hepatic poly(ADP ribose) polymerase (EC 2.4.2.30) activity as an indicator of DNA damage was measured in rats fed a low methionine, choline-devoid diet (MCD) for a 3-wk period. Additional groups of rats were either injected intraperitoneally (i.p.) with large doses of nicotinamide (NAM) or saline or fed the MCD diet without folic acid (MCFD). As a positive control, some rats were fed the MCD diet supplemented with methionine and choline (MCD + Met). In all groups of methyl donor-deficient rats and associated with increases in hepatic lipid levels, hepatic malondialdehyde concentrations were found to be increased. This observation is evidence for the occurrence of lipid peroxidation in methyl donor deficiency. Methyl donor deficiency was also associated with a significantly elevated hepatic poly(ADP ribose) polymerase activity in all groups of rats as compared to the positive control, suggesting a stimulation of DNA repair processes. The highest enzyme activity was observed in the MCD-NAM i.p. group.

Biosynthesis of catalytically active rat testosterone 5 alpha-reductase in microinjected Xenopus oocytes: evidence for tissue-specific differences in translatable mRNA.

The enzyme 4-ene-3-ketosteroid-5 alpha-oxidoreductase [5 alpha-reductase; 3-oxo-5 alpha-steroid delta 4-dehydrogenase, 3-oxo-5 alpha-steroid: (acceptor) delta 4-oxidoreductase, EC 1.3.99.5] plays a key role in androgen-dependent target tissues, where it catalyzes the conversion of testosterone to the biologically active dihydrotestosterone. The regulation of 5 alpha-reductase expression has not been studied at the molecular level as the enzyme is a membrane protein that is labile in cell-free homogenates. We developed a sensitive bioassay of the enzyme activity expressed in Xenopus oocytes microinjected with rat liver and prostate mRNA. After microinjection, incubation of intact oocytes in the presence of [3H]testosterone revealed the in ovo appearance of active 5 alpha-reductase. Polyadenylated RNA was fractionated by sucrose gradient centrifugation, and the enzymatic activity was shown to be encoded by a 1600- to 2000-base-pair fraction of hepatic poly(A)+ RNA. 5 alpha-Reductase mRNA was most efficiently translated when up to 80 ng of RNA was injected per oocyte. In the injected oocytes, 5 alpha-reductase mRNA was found to be a short-lived molecule (t1/2 = 2 hr), whereas its in ovo translatable 5 alpha-reductase protein exhibited stable enzymatic activity for over 40 hr. Moreover, the levels of translatable tissue-specific 5 alpha-reductase mRNAs as monitored in the Xenopus oocytes correlated with the variable 5 alpha-reductase activities in female rat liver, male rat liver, and prostate homogenates; the ratio of their specific activities was of 2500:630:1, respectively. Altogether, these results provide supporting evidence in favor of the transcriptional control of 5 alpha-reductase expression in rat tissues.

Ontogenically reculated expression of metallothionein and its messenger RNA in chick liver

By electrophoretic and immunological assay the concentration of hepatic metallothionein in new born chick liver was found to be ontogenically modulated, reaching a peak accumulation per gram liver in fourth day of hatching and declining below the detection limit after second week postnatal. The protein was undetectable upto second week of incubation in egg-embryonic stage. The concentration of metallothionein mRNA shows drastic change during first few days after hatching. The greatest accumulation of metallothionein mRNA was detected in the one day new born chicks, which declined rapidly there after, and reduced to a barely detectable level. Metallothionein was also detected in the in vitro translated product of one day neonatal chick hepatic poly(A+) RNA by S-cysteine labelling and immunoprecipitation. The naturally occurring new-born chick liver metallothionein was found to be a zinc-metallothionein and the concentration of hepatic zinc in new-born chick was found to undergo drastic modulation during development, unlike some other chick tissues. Endogenous zinc ion mobilization can thus play a significant role in the developmental regulation of chick metallothionein expression.

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2.

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.

Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase.

Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis.

Ovine β-lactoglobulin messenger RNA: Nucleotide sequence and mRNA levels during functional differentiation of the mammary gland

The nucleotide sequence of ovine beta-lactoglobulin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and primer extension products. Ovine beta-lactoglobulin mRNA consists of a 540 nucleotide coding region, flanked by 39 nucleotide 5' and 206 nucleotide 3' non-coding regions including a 20 nucleotide poly A tail. The deduced 180 amino acid sequence of pre-beta-lactoglobulin is in agreement with the previously published amino acid sequence of signal peptide and mature protein. Northern blot analysis of poly A+ RNAs from the lactating mammary glands of porcine, rabbit and rat species, allowed us to identify a homologous RNA to beta-lactoglobulin mRNA solely in the porcine species. We also detected a mRNA transcript of a size similar to that of beta-lactoglobulin mRNA in hepatic poly A+ RNA from female rat liver treated by estrogens. Furthermore, we have examined the levels of beta-lactoglobulin mRNA during the functional differentiation of the mammary gland and after hormonal stimulation. During the last third of pregnancy, the expression of beta-lactoglobulin gene is significantly more elevated than that of alpha s1- or beta-casein whose mRNA levels were found to change very slightly during this period. Both beta-lactoglobulin and casein mRNAs showed a rapid response and a wide range of change in response to cortisol treatment. However, there was a significant difference in the rate at which these processes occurred, suggesting that beta-lactoglobulin gene expression is regulated independently of the casein genes.

Induction of UDP-glucuronyl transferase mRNA in embryonic chick livers by phenobarbital

Administration of phenobarbital to chick embryos increased hepatic microsomal UDP-glucuroyltransferase activity some 25-fold. The large phenobarbital-induced increase of UDP-glucuronyltransferase activity was correlated to an equivalent increase of immunochemically measurable UDP-glucuronyltransferase protein. Poly(A +) RNA isolated from the livers of chick embryos treated with either phenobarbital or saline was translated in vitro. Immunochemical analysis of the translation products indicated that phenobarbital induced a 30-fold increase in UDP-GT mRNA. Fractionation of hepatic poly(A +) RNA from phenobarbital-treated chick embryos by sucrose density gradient centrifugation indicated that the size of the UDP-GT mRNA was 21S. These data show that phenobarbital induction of chick embryo liver UDP-glucuronyltransferase activity correlates with a similar large increase in the level of translatable mRNA for this enzyme.

Expression of rat NADPH-cytochrome P-450 reductase cDNA in Saccharomyces cerevisiae.

A full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats. Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences. Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions. Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction into Saccharomyces cerevisiae AH22 cells, they synthesized about 1 X 10(3) and 5 X 10(3) reductase protein molecules per cell, respectively. A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 X 10(5) cytochrome P-450MC hemoprotein and 1 X 10(4) reductase protein molecules per cell. The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochrome c reductase and cytochrome P-450MC-dependent 7-ethoxycoumarin O-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC. 7-Ethoxycoumarin O-deethylation activity in the cellular extract of AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG. In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells. These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed in S. cerevisiae cells.