[Discussion of grading method of small opacity profusion of pneumoconiosis on CT scans and the corresponding reference images].

Objective: To explore the CT grading method of small opacity profusion of pneumoconiosis, and draw up the corresponding CT reference film. Methods: In December 2019, Three hundred thirty-seven cases of pneumoconosis and suspected pneumoconiosis were examined by chest radiography and Computed Tomography (CT) in the same period. According to Diagnosis of Occupational Pneumoconiosis (GBZ 70-2015) , small opacity profusion of pneumoconiosis in each zone of lung was divided. On CT scans, it was divided into 5 grades of 0, 0+, 1, 2 and 3. Grade 0 corresponded to Sub-grade 0/- and Sub-grade 0/0 of Grade 0 in chest radiograph. Grade 0+ was equivalent to Sub-grade 0/1 of Grade 0. Grade 1, 2, 3 were equivalent to Grade 1, 2 and 3, respectively (including each sub-grade) . The CT image quality of each zone of lung was divided into 1 to 4 levels. Results of level 4 were not included in statistical analyses.Based on the results of small opacity profusion in each zone of lung, consistency analysis was performed between chest radiograph and CT. The selection method of reference films was developed. Based on the types and grades of small opacity, the final reference films were determined. Results: There were 1877 zones of lung with CT image quality from level 1 to 3, including 335 in upper right, 319 in middle right, 284 in lower right, 334 in upper left, 320 in middle left and 286 in lower left. The Kappa values of small opacity profusion in upper right zone, upper left zone, left middle zone, and lower left zone were all between 0.4-0.75. In middle right zone and lower right zone, they were all above 0.75.Among all 6 zones of lung, the diagnostic concordance rates between CT and chest radiograph were all above 80%.The corresponding CT reference films were proposed, including type p and q in Grade 2 and 3, type r in Grade 2, type s and t in Grade 0+ to 3. Conclusion: The CT grading method for small opacity profusion of pneumoconiosis is feasible, and the application value of its reference films needs to be further verified.

Edaravone protects from retinal injury through NF-κB in diabetic rats.

OBJECTIVE: To observe whether edaravone has a therapeutic and protective effect on retinal injury in diabetic rats through the nuclear factor-κB (NF-κB) pathway. MATERIALS AND METHODS: The Sprague-Dawley rat model of diabetes was established and divided into diabetes model group (model group) and edaravone treatment group (treatment group). Also, normal control group (control group) was set up. After successful modeling, the blood and retinal tissues of rats were collected. Then, the blood glucose content and serum interleukin-6 (IL-6) were detected. Antioxidant indexes, superoxide dismutase (SOD), and malondialdehyde (MDA), were detected via enzyme-linked immunosorbent assay (ELISA), while the number of corneal nerve fibers was observed microscopically. Moreover, the gene and protein expressions of SOD and NF-κB pathway in tissues were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting. RESULTS: The level of blood glucose in model group was increased compared with that in control group (p<0.05), indicating the successful modeling. The levels of tumor necrosis factor-α (TNF-α), IL-6, and IL-1 were significantly higher in model group than those in control group. In terms of the antioxidant indexes, the level of MDA in model group was significantly higher than that in other two groups, while the level of SOD in treatment group was significantly increased and close to that in control group. Besides, the number of corneal nerve fibers significantly declined in model group, while it was increased in treatment group but still lower than that in control group. According to the gene detection results, the mRNA expression of NF-κB p65 was markedly higher in model group than that in control group, and it was decreased in treatment group, while the mRNA expression of SOD showed the opposite trend. The protein expression of NF-κB p65 was remarkably higher in model group than in control group, was decreased in treatment group, and was close to that in control group, while the protein expression of SOD showed the opposite trend. CONCLUSIONS: Edaravone can affect the oxidation and antioxidation through the NF-κB signaling pathway, thereby exerting a therapeutic and protective effect on retinal injury in diabetic rats.

Application of levator veli palatini retropositioning combined with Buccinator myomucosal island flap for congenital cleft palate.

Congenital cleft palate causes a serious obstacle to children with regard to language and eating function. The aim of the current study was to examine the clinical application of a type of palatoplasty that has a reduced impact on the maxillary growth and good function in velopharyngeal competence. A total of 37 patients with cleft palate were treated with levator veli palatini retropositioning combined with Buccinator myomucosal island flap. The patients were successfully treated in the first phase and were followed up for 1-3 years. Speech intelligibility was satisfactory and no fistula occurred. In conclusion, the results suggested that levator veli palatini retropositioning combined with the Buccinator myomucosal island flap may restore normal anatomic structure and location of the levator veli palatini, obtain good velopharyngeal competence, and decrease the incidence rate thereof. Thus, levator veli palatini retropositioning combined with the Buccinator myomucosal island flap is a functional procedure for cleft palate repair.

Analysis of imprinted messenger RNA expression in deceased transgenic cloned goats.

Genomic imprinting is an important epigenetic mechanism that has vital effects on fetal growth and development. We observed the differences in four tissues (heart, spleen, liver, and kidney) from dead transgenic cloned goats using hematoxylin and eosin (H&E) staining. Eight imprinted genes in the tissues of dead transgenic cloned and normal goats were analyzed using reverse transcription polymerase chain reaction. H&E staining results from the abortion group indicated the lack of obvious morphological changes in heart and spleen tissues, while inflammatory cell infiltration and glomerular nephritis characteristics were observed in liver and kidney tissues, respectively. Compared to the control group, CDKN1C, H19, IGF2R, and SNRPN were significantly (P < 0.05) overexpressed in the heart tissue of the abortion group, while XIST was significantly reduced. In the liver tissues, CDKN1C and DLK1 expression decreased, while GNAS, H19, IGF2R, PEG3, and XIST expression increased significantly. In the spleen tissues, DLK1 expression increased, while GNAS, H19, IGF2R, PEG3, SNRPN, and XIST expression decreased. In the kidney tissues, CDKN1C, DLK1, GNAS, IGF2R, and PEG3 expression increased, while H19 and XIST expression decreased. The overall expression of imprinted genes was abnormal in different tissues of transgenic cloned goats, and the degree of abnormal genomic imprinting was more severe in the abortion group compared to the death and control groups. These results suggest that abnormal expression of imprinted genes may cause developmental defects in transgenic cloned goats. Moreover, abnormal epigenetic modifications may affect the reprogramming of transgenic donor cells.

Identification of transgenic cloned dairy goats harboring human lactoferrin and methylation status of the imprinted gene IGF2R in their lungs.

Dairy goat is a good model for production of transgenic proteins in milk using somatic cell nuclear transfer (SCNT). However, animals produced from SCNT are often associated with lung deficiencies. We recently produced six transgenic cloned dairy goats harboring the human lactoferrin gene, including three live transgenic clones and three deceased transgenic clones that died from respiratory failure during the perinatal period. Imprinted genes are important regulators of lung growth, and may be subjected to faulty reprogramming. In the present study, first, microsatellite analysis, PCR, and DNA sequence identification were conducted to confirm that these three dead kids were genetically identical to the transgenic donor cells. Second, the CpG island methylation profile of the imprinted insulin-like growth factor receptor (IGF2R) gene was assessed in the lungs of the three dead transgenic kids and the normally produced kids using bisulfite sequencing PCR. In addition, the relative mRNA level of IGF2R was also determined by real-time PCR. Results showed that the IGF2R gene in the lungs of the dead cloned kids showed abnormal hypermethylation and higher mRNA expression levels than the control, indicating that aberrant DNA methylation reprogramming is one of the important factors in the death of transgenic cloned animals.

Effect of Age on Energy Requirement for Maintenance and Growth of Dorper and Hu Crossbred F1 Ewes Weighing 20 to 50 kg.

This research aimed to define the energy requirement of Dorper and Hu Hybrid F1 ewes 20 to 50 kg of body weight, furthermore to study energy requirement changes with age and evaluate the effect of age on energy requirement parameters. In comparative slaughter trial, thirty animals were divided into three dry matter intake treatments (ad libitum, n = 18; low restricted, n = 6; high restricted, n = 6), and were all slaughtered as baseline, intermediate, and final slaughter groups, to calculate body chemical components and energy retained. In digestibility trial, twelve ewes were housed in individual metabolic cages and randomly assigned to three feeding treatments in accordance with the design of a comparative slaughter trial, to evaluate dietary energetic values at different feed intake levels. The combined data indicated that, with increasing age, the net energy requirement for maintenance (NEm) decreased from 260.62±13.21 to 250.61±11.79 kJ/kg(0.75) of shrunk body weight (SBW)/d, and metabolizable energy requirement for maintenance (MEm) decreased from 401.99±20.31 to 371.23±17.47 kJ/kg(0.75) of SBW/d. Partial efficiency of ME utilization for maintenance (km, 0.65 vs 0.68) and growth (kg, 0.42 vs 0.41) did not differ (p>0.05) due to age; At the similar condition of average daily gain, net energy requirements for growth (NEg) and metabolizable energy requirements for growth (MEg) for ewes during late fattening period were 23% and 25% greater than corresponding values of ewes during early fattening period. In conclusion, the effect of age upon energy requirement parameters in the present study were similar in tendency with previous recommendations, values of energy requirement for growth (NEg and MEg) for Dorper and Hu crossbred female lambs ranged between the NRC (2007) recommendation for early and later maturating growing sheep.

Hemodynamic changes of unexplained syncope patients in head-up tilt test.

The purpose of this study was to observe the hemodynamic changes of unexplained syncope patients in the head-up tilt test and their correlations with age and gender. Eighty-six patients with unexplained syncope were administered the basic head-up test and nitroglycerin provocation test with continuous monitoring and recording of electrocardiogram and blood pressure changes. Basic characteristics of the patients and their hemodynamic responses throughout the tests were analyzed. All 86 patients tolerated and completed the head-up test. Forty-nine (56.98%) of the patients displayed a positive reaction, 37 (43.02%) patients displayed a negative reaction. Patients were divided into groups as follows: Group A, age ≤ 35 years; Group B, age 36-45 years; and Group C, age ≥ 46 years. Older patients were more prone to chronotropic incompetence, and younger patients were more prone to an excessive increase in heart rate. Older age correlated with the occurrence of autonomic nerve reaction disorder and mixed vasovagal syncope, whereas younger age was related to the occurrence of vasodepressor type vasovagal syncope (P < 0.01). Gender did not significantly correlate with negative or positive head-up test results (P = 0.184). During the head-up test, younger patients mainly manifested an excessive heart rate increase, whereas older patients did not have significant heart rate changes. Analyzing the hemodynamic changes in the head-up test and studying the relationships between age, gender, and hemodynamic responses are crucial to determine etiologies of syncope and select appropriate treatment.

Hepatic DNA methylation modifications in early development of rats resulting from perinatal BPA exposure contribute to insulin resistance in adulthood.

AIMS/HYPOTHESIS: Perinatal exposure to bisphenol A (BPA), a widely distributed environmental endocrine disruptor, is associated with insulin resistance and diabetes in offspring. The underlying molecular mechanisms could involve epigenetics, as adverse effects induced by environmental exposure in early life are suggested through DNA methylation. In this study we sought to elucidate the relationship between perinatal BPA exposure and alteration of hepatic DNA methylation. METHODS: Pregnant Wistar rats were administered BPA (50 µg/kg/day) or corn oil by oral gavage throughout gestation and lactation. Variables associated with insulin resistance and hepatic DNA methylation were examined at postnatal week 3 and week 21 in male offspring. RESULTS: In BPA-treated offspring, serum insulin and HOMA-insulin resistance were increased, and the insulin sensitivity index and hepatic glycogen storage were decreased compared with controls at week 21. At week 3, none of these variables were significantly changed. However, hepatic global DNA methylation was decreased, accompanied by overexpression of DNA methyltransferase 3B mRNA at week 3. Meanwhile, perinatal exposure to BPA induced promoter hypermethylation and a reduction in gene expression of hepatic glucokinase. Moreover, increased promoter hypermethylation of Gck became more pronounced in BPA-treated offspring at week 21. CONCLUSIONS/INTERPRETATION: Abnormal DNA methylation in hepatic tissue precedes development of insulin resistance induced by perinatal BPA exposure. These findings support the potential role of epigenetics in fetal reprogramming by BPA-induced metabolic disorders.

Production of dairy goat embryos, by nuclear transfer, transgenic for human acid beta-glucosidase.

Expression of recombinant human lysosomal acid beta-glucosidase (hGCase) by a transgenic animal bioreactor, using somatic cell nuclear transfer (SCNT), would decrease the cost of producing this product. The objective was to establish an effective procedure to prepare hGCase transgenic donor cells and nuclear transfer (NT) embryos to produce hGCase protein in the Saanen dairy goat mammary gland. A mammary-specific expression vector for hGCase was constructed and transfected into HC-11 mammary epithelial cells for bioactivity analysis in vitro; mRNA transcripts and hGCase protein were correctly expressed in transfected HC-11 cells. The hGCase gene was then introduced into fetal fibroblasts (from dairy goats) to prepare competent transgenic donor cells. Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates and screened with PCR amplification and chromosomal counting (66.8%). Dairy goat cloned embryos were produced from these hGCase fetal cells by SCNT, the hGCase transgene was successfully detected in these embryos, and there were similar rates (P>0.05) of fusion (83.3% vs. 77.8%), cleavage (89.1% vs. 90.9%), and development to the morula/blastocyst stages (36.4% vs. 38.9%) between NT embryos using transgenic fetal fibroblasts and non-transfected control cells. Moreover, 98 well-developed reconstructed embryos derived from transgenic cells were transferred to 16 recipients; pregnancy was confirmed at 40 d in two goats. Therefore, we achieved functional expression of hGCase in mammary gland cells and normal development to Day 40 of cloned embryos carrying the hGCase gene.

CYP2D6 polymorphism in a Mexican American population.

BACKGROUND: Pharmacogenetic data are largely unavailable for Mexican Americans, despite being one of the largest populations in America. METHODS: The CYP2D6 genotype (n = 349) and dextromethorphan hydroxylation phenotype (n = 285) were studied in 380 Mexican American subjects from Los Angeles County. RESULTS: The allelic frequency was 22.8% for CYP2D6*2, 10.3% for CYP2D6*4, 7.4% for CYP2D6*10, 2.3% for CYP2D6*5, 1% for CYP2D6*XN (duplication), and <1% for CYP2D6*3 and CYP2D6*17. By using the published antimode for Caucasians, we identified nine subjects as poor metabolizers, an incidence of 3.2%. Of the eight poor metabolizers who were also genotyped, five either were homozygous for the CYP2D6*4 allele (4 cases) or had a combination of CYP2D6*4 and CYP2D6*5 alleles. The mean log(10) dextromethorphan/dextrorphan ratio was -2.47 for those classified as extensive metabolizers. The number of functional alleles among the extensive metabolizers correlated strongly with the phenotype, suggesting a gene-dose effect. CONCLUSION: Compared with previous reports on Caucasian populations, studies show that Mexican Americans appear to possess a lower rate of CYP2D6*4. Frequencies for the other alleles appear to be less divergent between the two groups. This genotypic pattern might be responsible for the lower rate for the poor metabolizer status, as well as for the faster enzyme activity in the extensive metabolizer subjects that was also reflected in our data.

Analysis of the CYP2D6 gene polymorphism and enzyme activity in African-Americans in southern California.

Despite its importance in drug metabolism and disease susceptibility, CYP2D6 activity and genetic polymorphism have rarely been investigated in African-American populations. In order to bridge this gap, we examined the genotype and phenotype of the enzyme in 154 African-American (AA) and 143 Caucasian (C) normal volunteers. AAs are significantly more likely to possess *17 and *5, but less likely to have *4. Overall, the two groups were similar in their CYP2D6 activity as measured with dextromethorphan as the probe (metabolic ratio 2.21 +/- 0.78 for AAs; 2.11 +/- 0.86 for Cs; t = 1.02, NS). Two of four AAs and six of seven Cs were classified as poor metabolizers and have two nonfunctioning alleles. CYP2D6 activity is determined by *17, *4, *5 and age in AAs (r2 = 0.33, f = 18.8, P < 0.001) and by *4 and *XN in Cs (r2 = 0.14, f = 10.8, P < 0.001). These results support previous findings demonstrating the importance of *17 in determining CYP2D6 activity in AAs.

Mallory bodies formed in proteasome-depleted hepatocytes: an immunohistochemical study.

Mallory bodies (MBs) are aggregates of proteins, principally cytokeratin proteins found in liver cells. They are also found in a few other cell types such as type II pneumocytes and trophoblasts. Studies on the liver thus far indicate that MBs are derived from hyperphosphorylated, heavily ubiquitinated proteins which have undergone conformational change. The aggregated protein may accumulate because of the failure of the proteasome to remove the altered proteins from the cytoplasm of liver cells. To investigate this possibility, the proteasomes were assessed immunohistochemically in individual liver cells of mice fed a drug which induced MB formation. To accelerate and enhance MB formation, cytochrome P450 2EI knockout mice were used. Proteasomes in individual cells were visualized by immunofluorescence using an antibody to a subunit of the proteasome (P25). The results showed that the groups of liver cells that had formed MBs were often partially depleted of proteasomes. These findings support the possibility that MBs formed as a result of the loss of the proteasome to remove misfolded cytokeratin proteins. Thus MBs may share their pathogenesis with other types of cellular inclusions seen where proteins aggregate in the cytoplasm due to mutation, misfolding, or loss of proteasomes.

Regulation of peroxisome proliferator activated receptor alpha-mediated pathways in alcohol fed cytochrome P450 2E1 deficient mice.

Fatty acids are substrates and inducers for cytochrome P450 2E1 (CYP2E1) and peroxisome proliferator activated receptor alpha (PPARalpha). Previously, we have shown that the ethanol-induced CYP2E1 expression in rat is accompanied by the inhibition of the expression of the PPARalpha gene and the reduction in polyunsaturated fatty acid content. To further analyze the effect of CYP2E1 and ethanol in PPARalpha-mediated fatty acid homeostasis, the expression of PPARalpha and retinoid x receptor alpha (RXRalpha) and their target genes was examined in ethanol fed CYP2E1 deficient mice. Our data demonstrated that the expression of PPARalpha and RXRalpha genes was activated in the livers of CYP2E1-null mice suggesting a compensatory effect for the absence of CYP2El. In addition, the expression of PPARalpha target genes, which included the liver fatty acid-binding protein, malic enzyme, and CYP4A1 genes, was induced indicating the activation of PPARalpha-mediated pathways in CYP2E1 deficient mice. Ethanol inhibited the expression of some of the PPARalpha target genes in wild-type mouse livers, and the inhibitory effect of ethanol was particularly prominent in the CYP2E1-null mice. Morphologically, centrilobular fat accumulation was detected in the ethanol fed CYP2E1-null mouse livers suggesting that inhibition of PPARalpha-mediated pathways might be responsible for the ethanol-induced fatty liver in CYP2El-null mice. In addition, the expression of CYP2E1 was not changed in the PPARalpha-null mice. These data suggest that CYP2E1 and ethanol can regulate PPARalpha-mediated fatty acid homeostasis. CYP2E1-induced lipid peroxidation might play a major role in lipid metabolism, PPARalpha only becomes important when the CYP2E1 level is low and polyunsaturated fatty acids increase.

Dexamethasone enhances mallory body formation in drug-primed mouse liver.

In a clinical study in which patients with alcoholic hepatitis were treated with prednisone for 1 month, posttreatment liver biopsies showed diminished inflammation, but Mallory bodies were not diminished. This suggested that steroid treatment may reduce inflammation by inhibiting NFkappaB activation. Sparing of Mallory bodies suggests that NFkappaB activation may not be involved mechanistically in Mallory body formation. To test this idea, we induced Mallory body formation in drug-primed mice with or without dexamethasone treatment. As predicted, dexamethasone decreased NFkappaB activation; however, Mallory body formation was increased. Surprisingly, TNFalpha and iNOS, which normally increase as a result of NFkappaB activation, were upregulated by the dexamethasone treatment. It was concluded that NFkappaB activation is not involved in Mallory body formation. Despite this, induced increases in TNFalpha, iNOS, c-jun/API and c-myc expression indicate that oxidative stress is likely involved in Mallory body formation.

Identification of retinoic acid-responsive elements on the HNF1alpha and HNF4alpha genes.

Hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha are liver-selective transcription factors and are essential for hepatocyte differentiation. This study demonstrates that HNF1alpha as well as HNF4alpha genes contain a direct repeat with a space of one nucleotide (DR1)-retinoic acid (RA) response element that can be bound and regulated by RA and retinoid x receptor alpha (RXRalpha) complex. Transient transfection experiments showed that RA increased the promoter activity of the HNF1alpha and HNF4alpha genes in Hep3B cells. Overexpression of RXRalpha further enhanced the activities of both genes. Two putative RXRalpha binding sites on the HNF1alpha (-295 to -276) and HNF4alpha (-418 to -399) genes have been characterized. By transient transfection, both sites positively responded to RA, and overexpression of RXRalpha in Hep3B cells increased the regulatory effect. Gel mobility shift assay demonstrated that these two DR-1 sites could be bound by RXRalpha specifically. These data suggest that the differentiation effect of RA on hepatocyte may be due to direct interaction of RXRalpha with the RA-responsive elements on the HNF1alpha and HNF4alpha genes.

Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice.

Retinoid x receptor alpha (RXRalpha) serves as an active partner of peroxisome proliferator-activated receptor (PPARalpha). In order to dissect the functional role of RXRalpha and PPARalpha in PPARalpha-mediated pathways, the hepatocyte RXRalpha-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXRalpha and PPARalpha deficiency are different in several aspects. At the basal untreated level, RXRalpha deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPARalpha-null mice. Fasting-induced PPARalpha activation was drastically prevented in the absence of hepatocyte RXRalpha. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXRalpha. Hepatocyte RXRalpha deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXRalpha-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXRalpha. These data suggested that in comparison to PPARalpha, hepatocyte RXRalpha has its unique role in lipid homeostasis and that the effect of RXRalpha, -beta, and -gamma is redundant in certain aspects.

Fatty acyl-CoAs inhibit retinoic acid-induced apoptosis in Hep3B cells.

Retinoic acid (RA) induces apoptosis in Hep3B human hepatoma cells. 9-Cis-RA (c-RA) had a similar effect as all-trans-RA (t-RA) in inducing cell death in Hep3B cells. RA-induced Hep3B-cell death was associated with inhibited expression of the hepatocyte nuclear factor 4 (HNF-4) gene. Palmitoyl-CoA ((C16:0)-CoA), the reported HNF-4 ligand, prevented RA-induced apoptosis. The effect of (C16:0)-CoA was specific, since palmitic acid and co-enzyme A had no effect in preventing RA-induced apoptosis. Bovine serum albumin (BSA) also prevented RA-induced apoptosis. However, in contrast to BSA, which induced cell growth, (C16:0)-CoA alone had no effect on cell growth. Investigating the possible role of HNF-4 in apoptosis, the reported HNF-4 antagonist (C18:0)-CoA was employed, and it also prevented RA-induced apoptosis. By transient transfection, overexpression of HNF-4 did not prevent RA-induced apoptosis. The induction and prevention of apoptosis caused by RA and (C16:0)-CoA were associated, respectively with the induction and inhibition of the expression of transforming growth factor beta (TGFbeta), which is known to play a role in apoptosis. Furthermore, RA and (C16:0)-CoA can regulate AP-1, which is a key regulator of the TGFbeta gene. Our data indicate that fatty acyl-CoAs can prevent RA-induced apoptosis and that TGFbeta, rather than HNF-4, may play a role in these regulatory processes. Our data also suggest that (C16:0)-CoA and (C18:0)-CoA are not the agonist and antagonist for HNF4, respectively in the Hep3B cell system.

Hepatocyte-specific mutation establishes retinoid X receptor alpha as a heterodimeric integrator of multiple physiological processes in the liver.

A large number of physiological processes in the adult liver are regulated by nuclear receptors that require heterodimerization with retinoid X receptors (RXRs). In this study, we have used cre-mediated recombination to disrupt the mouse RXRalpha gene specifically in hepatocytes. Although such mice are viable, molecular and biochemical parameters indicate that every one of the examined metabolic pathways in the liver (mediated by RXR heterodimerization with PPARalpha, CARbeta, PXR, LXR, and FXR) is compromised in the absence of RXRalpha. These data demonstrate the presence of a complex circuitry in which RXRalpha is integrated into a number of diverse physiological pathways as a common regulatory component of cholesterol, fatty acid, bile acid, steroid, and xenobiotic metabolism and homeostasis.

Effect of cigarette smoking on coumarin metabolism in humans.

Extant data, mostly from studies in vitro, suggest that coumarin and nicotine are both metabolized by CYP2A6, a cytochrome P450 isozyme. In order to investigate this issue further, the activity of this enzyme in vivo was measured in 37 non-smokers and 37 smokers using coumarin (2.0 mg, PO) as the metabolic probe. The percentage of coumarin metabolized to 7-hydroxycoumarin in 8 h was measured in urine by high-pressure liquid chromatography. There was more than 10-fold variability in coumarin metabolism in both groups. Coumarin metabolism was significantly reduced in smokers (46.6 +/- 4.4%) as compared to non-smokers (66.4 +/- 3.5%; p < or = .001). The results support previous in vitro findings that both coumarin and nicotine are metabolized, at least in part, by a common pathway, which most likely is CYP2A6.

Role of CYP2E1 in the pathogenesis of alcoholic liver disease: modifications by cAMP and ubiquitin-proteasome pathway.

The ethanol inducible isoform of cytochrome P450, CYP2E1, may play a role in ethanol-induced liver injury. Therefore, the factors which govern CYP2E1 degradation and turnover were investigated. These factors include cAMP, ubiquitin, proteasomal enzymes and CYP2E1 mRNA. Rats fed ethanol or pair-fed isocaloric dextrose were pair-fed with rats fed ethanol or dextrose treated with cAMP for 2 months. The liver pathology, regenerative activity, fatty acid composition, NFkappaB activation, ubiquitin conjugates and proteasomal enzymes were measured as were the apoprotein levels of CYP2E1, CYP3A, CYP4A and mRNA levels for CYP2E1 and ubiquitin expression. The results showed, that the cAMP treatment ameliorated the increase liver fat storage and changes in the fatty acid composition in the livers of ethanol fed rats. Other histologic features of alcoholic liver disease were not changed. Western blot quantitation showed that the amount of ubiquitin and ubiquitin conjugates were markedly reduced by ethanol treatment. Similarly, ethanol decreased the level of ubiquitin mRNA. cAMP ameliorated the inhibition of the proteasomal enzyme proteolysis caused by ethanol feeding. The ethanol-induced increase in the CYP2E1 protein was partially inhibited by cAMP treatment. cAMP treatment decreased CYP2E1 mRNA levels in both ethanol-fed and pair fed control rats. Likewise NFkappaB activation was not increased by ethanol but cAMP reduced the level of NFkappaB activation. CAMP treatment also reduced CYP4A but not CYP3A. The results support the concept that cAMP treatment partially protects the liver from ethanol-induced fatty liver by reducing CYP2E1 induction through cAMP's effects on CYP2E1 synthesis.