Real-World Efficacy of Azelaic Acid 15% Gel for the Reduction of Inflammatory Lesions of Rosacea.

Approximately 16 million Americans have rosacea, an inflammatory cutaneous disorder with central facial erythema, papules, pustules, telangiectasia, flushing, and swelling being among the more commonly recognized features. Overexpression of cathelicidin peptide LL-37 has been implicated in the pathophysiology of rosacea. Azelaic acid has been found to inhibit the pathologic expression of cathelicidin, as well as the hyperactive protease activity that cleaves cathelicidin into LL-37. Given these findings, a small prospective, open-label, interventional trial was undertaken to assess the effects of azelaic acid 15% gel on inflammatory lesions of papulopustular rosacea in a real-world setting. Use of azelaic acid was associated with a significant reduction in inflammatory lesions, which persisted beyond the active treatment phase. Overall, azelaic acid 15% gel is an appropriate initial topical therapy for the treatment of moderate facial rosacea.

Coordinate polypeptide expression during hepatocarcinogenesis in male F-344 rats: comparison of the Solt-Farber and Reddy models.

The Solt-Farber resistant hepatocyte (RH) and Reddy (dietary peroxisome proliferator) hepatocarcinogenesis protocols were utilized to induce both preneoplastic and neoplastic nodules in male F-344 rats. Total cellular polypeptides from normal liver, ciprofibrate (CP)-induced and RH nodules were analyzed for both qualitative and quantitative changes using computer-assisted, high resolution two-dimensional polyacrylamide gel electrophoresis. Approximately 800-1000 cytosolic and 1000-1200 particulate polypeptides were readily separated and detected using an ultrasensitive silver stain. The two-dimensional polyacrylamide gel electrophoresis patterns were very similar for each tissue with respect to both the number of polypeptides detected and the overall patterns. Three cytosolic polypeptides, E, 6.90/47; F, 6.90/46; and G, 6.50/28 (designated pI/Mr X 10(-3], and two particulate polypeptides, B, 5.90/43; and D, 5.70/21; were detected in CP nodules but not in normal liver. Polypeptides B and D were also detected in RH nodules. No qualitative polypeptide differences were detected among the individual preneoplastic or individual neoplastic CP nodules or between preneoplastic and neoplastic CP nodules. Numerous quantitative changes in both known markers for hepatocarcinogenesis and in as yet unidentified polypeptides were noted. In RH nodules the Ya subunit of glutathione-S-transferase B (GST-B) and the Yb subunit of GST-A were increased 2-4-fold as compared to normal liver or in replicating liver following a 70% partial hepatectomy, while in CP nodules the Yb subunit was unaltered and the Ya subunit increased 4-fold as compared to normal. The Yp subunits of GST-P were increased from almost nondetectable levels in normal liver to one of the most abundant cytosolic polypeptides in RH nodules. In contrast, the Yp subunits were not detected in any of the CP nodules either on the two-dimensional polyacrylamide gel electrophoresis gels themselves or following Western transfer and immunoblot analysis with antibody against GST-P. Two additional polypeptide spots, which may represent Yc charge shift variants, appeared at the same molecular weight as the constitutively expressed Yc subunit of GST-B but shifted one charge unit each toward the acidic region in CP nodules. DT-diaphorase which was increased 2-3-fold in RH nodules was unaltered in CP nodules. In addition to these changes in known markers, 34 (22 cytosolic and 12 particulate) polypeptides were significantly increased while 27 (12 cytosolic and 15 particulate) polypeptides were decreased during CP-induced hepatocarcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of common patterns of polypeptide synthesis and phosphorylation by calcium and 12-O-tetradecanoylphorbol-13-acetate in mouse epidermal cell culture.

Terminal differentiation can be induced in cultured basal cells by either increasing the Ca2+ level in the medium from 0.05 to 1.4 mM or by exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). If Ca2+ and TPA act by a common mechanism, then a common pattern of protein synthesis and/or phosphorylation would be expected. Computer-assisted analysis of radioactively labeled polypeptides separated by two-dimensional-polyacrylamide gel electrophoresis was utilized to study protein synthesis and phosphorylation. Within 1 h of increasing the Ca2+ level in the medium, the synthesis of 57 polypeptides was altered by 2-fold or more. Similarly, exposure to TPA for 1 h affected the synthesis of 106 polypeptides. Sixteen polypeptides were affected by both Ca2+ and TPA; the synthesis of nine was increased and seven was decreased, with changes in the same direction for both effectors. By 4 h, the synthesis of 32 polypeptides was similarly modulated by both Ca2+ and TPA. Only one polypeptide which was increased at 1 h was still elevated at 4 h. These results suggest that a common dynamic program of protein synthesis, likely to be related to terminal keratinocyte differentiation, is induced by both Ca2+ and TPA. Overall phosphorylation of epidermal proteins was increased after 30 min of TPA treatment, but was not increased by Ca2+ at this time. Keratin polypeptides were heavily phosphorylated in low Ca2+ medium, but the level or pattern of phosphorylation of these proteins was not altered by either Ca2+ or TPA. Although phosphorylation of a minor polypeptide (pI 5.1/Mr 45,000) was increased 2-3-fold by both Ca2+ and TPA, most of the specific protein phosphorylation changes induced in keratinocytes by Ca2+ and TPA appear to be unique. Thus, if protein phosphorylation is an early signal for epidermal differentiation by each effector, only a single apparent common substrate is involved and multiple kinases are activated. Alternatively, substrate specificity of a single kinase may be differentially altered by each effector.

Comparison of gene expression in preneoplastic and neoplastic rat liver to adult, fetal, regenerating, and tumor-promoted liver.

Computer-assisted analysis was performed on the in vitro translation products of polyadenylated RNA samples isolated from normal adult Fischer rat liver and from preneoplastic and neoplastic rat liver samples which were generated by the Solt Farber technique (Solt, D. and Farber, E., Nature 263:701-703, 1976). The vast majority of the differences in translation products observed throughout the progressive development of hepatocellular carcinoma was quantitative in nature. Importantly, this quantitative heterogeneity first became prevalent at the very early preneoplastic stage of hepatoma formation. Only 3 consistent qualitative alterations in translation products were observed to be associated with the hepatocarcinogenesis process. The appearance of two new polypeptides of molecular weight and isoelectric point of 32/5.2 and 43/5.1 appeared to be related to an early preneoplastic event in hepatoma development and the transition from a preneoplastic to a neoplastic state, respectively. Importantly, these new polypeptides were not observed in the in vitro translation products generated from fetal or regenerating liver samples or from liver samples which were chronically treated with phenobarbital or terachlorodibenzo-p-dioxin. One translation product (located at 35/6.6) of normal adult, fetal, and regenerating liver RNA samples was undetected in all preneoplastic, neoplastic, phenobarbital-, and terachlorodibenzo-p-dioxin-treated liver RNA translation products. The possibility exists that the specific loss of this gene product may promote the development of the transformed phenotype.

Altered polypeptide expression associated with neoplastic transformation of Syrian hamster cells by bisulfite.

Neoplastic transformation of Syrian hamster fetal cells by bisulfite is associated with qualitative and quantitative polypeptide changes. Amino acid-labeled [14C]polypeptides from neoplastic and nontransformed parental fetal cells were separated by two-dimensional gel electrophoresis and analyzed by computerized microdensitometry of autoradiographic patterns. Approximately 1000 polypeptides from parental fibroblasts at population doublings ranging from 4 to 20, and those from colony-derived malignant cell lines were compared. Most were identical. Seven malignant lines exhibited 4 qualitative polypeptide changes: 2 polypeptides had shifted slightly to the acidic side, 1 new polypeptide was observed, and 1 polypeptide was absent. The transformed bisulfite lines differed quantitatively from control cells in that 10-25% and 2-4% of the polypeptides exhibited differences in expression greater than 2- and 4-fold, respectively. Furthermore, there were 21 specific polypeptides with coordinate quantitative changes in all transformed lines. Because bisulfite at neutral pH fails to induce any significant DNA changes at concentrations that cause transformation, polypeptides expressed immediately or 48 h after bisulfite treatment were compared to those of non-treated controls, and no differences were found. Even though bisulfite does not induce detectable DNA damage or early post-treatment changes in polypeptide expression, a consistent set of qualitative and quantitative changes were observed after transformation. The qualitative polypeptide changes found in the bisulfite-induced malignant lines were similar to those seen in a benzo(a)pyrene-induced malignant line. This suggests that there is a convergence of pathways responsible for carcinogenesis independent of the nature of initiation.

Sequential analysis of chemically induced hepatoma development in rats by two dimensional electrophoresis.

Using the Solt-Farber hepatocarcinogenesis model, a large population of preneoplastic and neoplastic nodules were induced in male Fischer 344 rats. Total cellular polypeptides from normal liver and individual preneoplastic and neoplastic nodules were analyzed for both qualitative and quantitative changes using computer assisted high resolution two-dimensional electrophoresis. Approximately 800-1000 cytosolic and 1200-1400 membrane associated polypeptides were readily separated and detected using an ultrasensitive silver stain. The polypeptide patterns were remarkably similar for each tissue and only four qualitative polypeptide differences were noted. One cytosolic polypeptide, 6.8/57 (designated pl/Mr X 10(-3), and three membrane associated polypeptides, 6.25/41, 6.75/24, and 6.05/21, were expressed in both preneoplastic and neoplastic nodules but not in normal liver. No qualitative polypeptide differences were detected among the individual preneoplastic or individual neoplastic nodules or between preneoplastic and neoplastic nodules. Numerous quantitative changes in both known markers for hepatocarcinogenesis and in as yet unidentified polypeptides were noted. In particular, the Ya subunit of glutathione S-transferase B, the Yb subunit of glutathione S-transferase A, as well as the three isoelectric point variants of the Yp subunit of glutathione S-transferase P were increased 2-, 4-, and 7-fold, respectively, in preneoplastic and neoplastic nodules. Whereas DT-diaphorase was increased 2-3-fold in hyperplastic nodules as compared to normal liver, no differences in the expression of albumin were noted. Although no differences were observed in the expression of aldehyde dehydrogenase in preneoplastic and neoplastic nodules, polypeptide b (6.9/54) was shifted slightly toward the basic region in normal liver. alpha-Fetoprotein was not detected in either preneoplastic or neoplastic nodules. In addition to these changes in known markers, comparison of 500-800 cytosolic and 750-1000 membrane associated polypeptides showed that roughly 4-10% of the polypeptides were undergoing quantitative changes of at least 4-fold during these stages of hepatocarcinogenesis. Thirty (10 cytosolic and 20 membrane) polypeptides were significantly down-regulated while 22 (7 cytosolic and 15 membrane) polypeptides were up-regulated in both preneoplastic and neoplastic nodules. In all cases the direction and magnitude of change were the same in both preneoplastic and neoplastic nodules with the exception of three polypeptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered responsiveness of rat liver epithelial cells to transforming growth factor beta 1 following their transformation with v-raf.

The effects of transforming growth factor beta (type 1) (TGF-beta 1) on DNA synthesis, cell proliferation, and protein synthesis were examined in a series of v-raf-transformed rat liver epithelial (RLE) cells, which exhibit a range of transformed phenotypes. All of the transformed cells were relatively resistant to the growth-inhibitory effects of TGF-beta 1, compared to normal RLE cells and control cells infected with a helper virus. The more tumorigenic cell lines had very few surface receptors for TGF-beta 1 and showed no increase in the secretion of a number of specific proteins, including fibronectin, following TGF-beta 1 treatment. In contrast, the more normal-looking, less tumorigenic v-raf-transformed cells bound similar amounts of TGF-beta 1 as normal RLE and control cells and showed a similar pattern of TGF-beta 1-stimulated protein secretion. These findings suggest that the effects of TGF-beta 1 on cell proliferation and on the expression of certain secreted proteins are mediated through different mechanisms. Following transformation of RLE cells with v-raf, the signalling pathways controlling TGF-beta 1 growth inhibition are perturbed, while those involved in regulating the synthesis of certain proteins may remain intact. Thus, the escape from the various distinct biological effects of TGF-beta 1 may be an important stage in the progression of neoplastic transformation of RLE cells in vitro.

Kinetics of N- and C-hydroxylations of 2-acetylaminofluorene in male Sprague-Dawley rat liver microsomes: implications for carcinogenesis.

The metabolism of 2-acetylaminofluorene (AAF) has been studied in male Sprague-Dawley rat liver microsomes over a concentration range of 0.02 to 300 microM, and kinetic parameters have been determined for five oxidative pathways. The N-hydroxylation of AAF was best described by a single enzyme system with a mean Km of 0.033 microM and a mean Vmax of 3.63 pmol/mg/min. Pretreatment of animals with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused a marked induction of N-hydroxylase activity while phenobarbital had no effect. Biphasic kinetics for the 7-hydroxylation of AAF were observed in both control and TCDD- and phenobarbital-induced microsomes. The high-affinity Km [0.051 +/- 0.015 (S.E.) microM; n = 3] in control microsomes was 3 orders of magnitude lower than the low-affinity Km (103 +/- 16 microM; n = 3) indicating that each isoenzyme predominated at vastly different substrate concentrations. The mean Vmax values for the low- and high-affinity enzymes were 3.5 and 1351 pmol/mg/min, respectively. TCDD pretreatment markedly induced the activity of the low-capacity enzyme and reduced the activity of the high-capacity enzyme. Phenobarbital caused a significant induction of both enzyme pathways. Biphasic kinetics were also observed for the 5-, 3-, and 1-hydroxylations of AAF in control and phenobarbital-induced microsomes, but in TCDD-pretreated microsomes only 1-hydroxylation exhibited biphasic kinetics. TCDD caused a marked induction of these metabolic pathways while phenobarbital had no effect. Nonclassical kinetics were observed for the 9-hydroxylation of AAF, and at high substrate concentrations detoxification via this pathway and 7-hydroxylation predominated. However, at low concentrations, metabolic activation of AAF via N-hydroxylation was a major pathway. These data indicate that multiple forms of cytochrome P-450 are involved in AAF metabolism and that the balance between metabolic activation and detoxification of this substrate is dependent on both concentration and previous exposure to inducers.

Isolation of preneoplastic rat liver cells by centrifugal elutriation and binding to asialofetuin.

Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model). The isolation procedure involved, after collagenase dispersion of the liver, separation of the hepatocytes into small- and large-cell fractions by centrifugal elutriation, and subsequent selection of cells deficient in asialoglycoprotein receptor(s) by plating onto asialofetuin (ASF)-coated plates. The number of cell surface binding sites for the asialoglycoprotein receptor was measured with both asialoorosomucoid and ASF as ligands. There was a 50% reduction of binding sites for both ligands in the original cell suspensions obtained from preneoplastic livers. The reduction in receptor binding sites was most pronounced in the large cell fraction (less than or equal to 30% of control value) after separating the original cell suspension by elutriation into small and large cell fractions. Immunohistochemical studies showed a lack of asialoglycoprotein receptor in preneoplastic (i.e., hyperplastic foci) areas. These areas were entirely super-imposable with glucose-6-phosphatase-deficient areas and partially overlapped the gamma-glutamyltranspeptidase-positive areas in serial liver sections. The attachment of preneoplastic hepatocytes to ASF-coated tissue culture dishes was greatly impaired, and the number of gamma-glutamyltranspeptidase-positive cells on the ASF dishes was reduced to less than 7% as compared to 45 to 70% on the collagen-coated plates. Thus, the lack of asialoglycoprotein (asialofetuin) surface receptors and the increased size of the early preneoplastic hepatocytes are characteristics that can be used to separate the preneoplastic cell population from normal liver cells.

Low frequency of p53 gene mutation in tumors induced by aflatoxin B1 in nonhuman primates.

Aflatoxin B1 has been suggested as a causative agent for a G to T mutation at codon 249 in the p53 gene in human hepatocellular carcinomas from southern Africa and Qidong in China. To test this hypothesis, nine tumors induced by aflatoxin B1 in nonhuman primates were analyzed for mutations in the p53 gene. These included four hepatocellular carcinomas, two cholangiocarcinomas, a spindle cell carcinoma of the bile duct, a hemangioendothelial sarcoma of the liver, and an osteogenic sarcoma of the tibia. None of the tumors showed changes at the third position of codon 249 by cleavage analysis of the HaeIII enzyme site at codon 249. A point mutation was identified in one hepatocellular carcinoma at the second position of codon 175 (G to T transversion) by sequencing analysis of the four conserved domains (II to V) in the p53 gene. These data suggest that mutations in the p53 gene are not necessary in aflatoxin B1 induced hepatocarcinogenesis in nonhuman primates. The occurrence of mutation in codon 249 of the p53 gene in selective samples of human hepatocellular cancers may indicate involvement of environmental carcinogens other than aflatoxin B1 or that hepatitis B virus-related hepatitis is a prerequisite for aflatoxin B1 induction of G to T transversion in codon 249.

Alterations of tumor suppressor genes and allelic losses in human hepatocellular carcinomas in China.

Aflatoxin B1 has been suggested as a causative agent for a G to T mutation at codon 249 in the p53 gene in human hepatocellular carcinomas (HCC) from southern Africa and Qidong in China. The objective of the present work was to test the hypothesis that exposure to aflatoxin B1 either alone or coincident with other environmental carcinogens might be associated with allelic losses occurring during development of human hepatocarcinogenesis in China. The HCCs were obtained from two different areas in China: Qidong, where exposure to hepatitis B virus (HBV) and aflatoxin B1 is high; and Beijing, where exposure to HBV is high but that of aflatoxin B1 is low. We analyzed the tumors for mutations in the p53 gene and loss of heterozygosity for the p53, Rb, and APC genes and at marker loci on chromosomes 4, 13, and 16. Frequencies of mutation, loss, and aberration (mutation and loss) of the p53 gene in 25 HCCs from Qidong were 60, 58, and 80%, respectively. The frequencies in 9 HCCs from Beijing were 56, 57, and 78%. However, the frequency of a G to T transversion at codon 249 in HCCs from Qidong and Beijing were 52 and 0%, respectively. These data indicate that mutation and/or loss of heterozygosity in the p53 gene, independent of the 249 mutation, play a critical role in the development of hepatitis B virus-associated HCCs in China. Loss of the Rb and APC genes was observed in 44 and 7% of HCCs from Qidong, respectively. Allelic losses on chromosome 4 and especially on chromosome 16 were frequent in HCCs from Qidong but were not observed in HCCs from Beijing, while loss of heterozygosity on chromosome 13 occurred at similar frequency in both Qidong and Beijing. These results show a distinct difference in the pattern of allelic losses between HCCs in Qidong and Beijing and suggest that aflatoxin B1 and/or other environmental carcinogens may contribute to this difference.

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo.

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.

Secreted and cellular polypeptide patterns of MCF-7 human breast cancer cells following either estrogen stimulation or v-H-ras transfection.

The polypeptide patterns of MCF-7 human breast cancer cells (MCF-7gpt) and a stably v-H-ras-transfected subclone (MCF-7ras) have been analyzed following estradiol treatment. Since both estradiol and v-H-ras transfection increase tumorigenicity of MCF-7 cells, this study was designed to ascertain if specific changes in polypeptides were common in both treatments. Separation of cellular and secreted polypeptides was accomplished by 2-dimensional polyacrylamide gel electrophoresis, and the consequent patterns were analyzed with computer assistance. Estradiol treatment of the MCF-7gpt cells reduced the number of differences found in the polypeptide patterns between MCF-7gpt and MCF-7ras. Twelve cellular polypeptides were consistently modulated by either estradiol or v-H-ras, with four polypeptides clearly affected in the same way by both treatments. Polypeptides Gchc-0845 (Mr 54,000, pI 6.9) and Gchc-0902 (Mr 52,000, pI 6.3) were suppressed by estradiol and v-H-ras, while Gchc-1240 (Mr 34,000, pI 4.4) and Gchc-1396 (Mr 23,000, pI 5.3) were induced by estradiol and v-H-ras. Sixteen secreted polypeptides were altered by at least 2-fold subsequent to estradiol treatment or v-H-ras transfection. Transfection with v-H-ras had a greater effect than estradiol, stimulating the secretion of eight polypeptides and suppressing the secretion of seven polypeptides compared to estradiol which increased secretion of five polypeptides and decreased secretion of an additional three polypeptides, respectively. Synergistic effects by estradiol and v-H-ras were noted for three polypeptides. The secretion of Gcls-175 (Mr 50,000, pI 5.7) and Gcls-320 (Mr less than 14,000, pI 3.6, p-S2) was increased, while the secretion of Gcls-112 (Mr 76,000, pI 6.9) was decreased. Opposing effects of estradiol and v-H-ras were seen for seven polypeptides including the Mr 48,000 derivative of the Mr 52,000 protein (cathepsin D). These studies support the possibility that an extremely few, but specific polypeptides are regulated in association with quite diverse tumorigenic stimuli in MCF-7 human breast cancer cells.

Dual functions of E2F-1 in a transgenic mouse model of liver carcinogenesis.

Deregulation of E2F transcriptional control has been implicated in oncogenic transformation. Consistent with this idea, we recently demonstrated that during hepatocarcinogenesis in c-myc/TGFalpha double transgenic mice, there is increased expression of E2F-1 and E2F-2, as well as induction of putative E2F target genes. Therefore, we generated transgenic mice expressing E2F-1 under the control of the albumin enhancer/promoter to test the hypothesis that E2F family members may contribute to liver tumor development. Overexpression of E2F-1 resulted in mild but persistent increases in cell proliferation and death during postnatal liver growth, and no increases in hepatic regenerative growth in response to partial hepatectomy. Nevertheless, from 2 months postnatally E2F-1 transgenic mice exhibited prominent hepatic histological abnormalities including preneoplastic foci adjacent to portal tracts and pericentral large cell dysplasia. From 6 to 8 months onward, there was an abrupt increase in the number of neoplastic nodules ('adenomas') with 100% incidence by 10 months. Some adenomas showed evidence of malignant transformation, and two of six mice killed at 12 months showed trabecular hepatocellular carcinoma. Endogenous c-myc was up-regulated in the early stages of E2F-1 hepatocarcinogenesis, whereas p53 was overexpressed in the tumors, suggesting that both E2F-1-mediated proliferation and apoptosis are operative but at different stages of hepatocarcinogenesis. In conclusion, E2F-1 overexpression in the liver causes dysplasia and tumors and suggests a cooperation between E2F-1 and c-myc oncogenes during liver oncogenesis.

The high transforming potency of erbB-2 and ret is associated with phosphorylation of paxillin and a 23 kDa protein.

Two-dimensional gel maps of proteins phosphorylated by the epidermal growth factor receptor (EGFR) and erbB-2 kinases were obtained, to investigate the molecular basis of the different biological properties of these two molecules. Several proteins were phosphorylated by EGFR or erbB-2 with different stoichiometry. Differences were either quantitative or qualitative. In NIH3T3 cells, erbB-2 is 100-fold more transforming than EGFR. In the same cell line several proteins were preferentially phosphorylated by erbB-2, as compared to EGFR. To identify which of these substrates might be directly involved in mitogenic signaling, we obtained two-dimensional maps of proteins phosphorylated on tyrosine by EGFR/ret and an EGFR/erbB-2TK chimeric receptors. Both these chimerae behaved indistinguishably from erbB-2 in a number of bioassays and potently transformed NIH3T3 cells. Paxillin and a 23 kDa substrate were invariably phosphorylated to higher stoichiometry whenever potent mitogenic and transforming signals were activated. We propose that paxillin and the 23 kDa substrate are important elements in the erbB-2 and ret-activated mitogenic and transforming signaling.

Derivatives of beta-adrenergic antagonists. N-Nitrosopropranolol and N-hydroxypropranolol and its aldonitrone.

Potential precursors to chemically reactive species derived from the beta-adrenergic antagonist propranolol were synthesized and tested for mutagenicity in the Ames Salmonella assay. N-Hydroxypropranolol (1), the corresponding aldonitrone, 3-(1-naphthoxy)-2-hydroxypropionaldehyde N-isopropylnitrone (2), and N-nitrosopropranolol (3) were prepared and tested. N-Hydroxypropranolol (1) was obtained by direct alkylation of 3-(1-naphthoxy)-1-bromo-2-propanol with N-isopropylhydroxylamine and isolated as its neutral oxalate or HBr salt. The aldonitrone (2) was obtained by mercuric oxide oxidation of the hydroxylamine. N-Nitrosopropranolol (3) was prepared by treating propranolol with nitrous acid. None of the compounds was mutagenic in the Ames assay with Salmonella typhimurium TA-98 and TA-100 strains, either in the absence or in the presence of the S-9 liver fraction from Arochlor 1254 treated rats. None of the compounds was significantly toxic to the bacteria, except for slight toxicity of the oxalate salt of 1.

Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis.

The high resolving power of two-dimensional polyacrylamide gel electrophoresis 2D-PAGE and its full analytical and preparative potential have been described with special emphasis on reproducibility and standardization of protein spot patterns, enhanced protein detection sensitivity, and computer analysis database development. New methodologies for peptide mass fingerprinting, peptide, sequence, and fragmentation tagging have been highlighted. Major challenges associated with 2D-PAGE/mass spectrometric protein sequencing were outlined which need to be addressed in the future, including sample enrichment, use of alternative gel matrices, improvements in separation systems interfaced directly to the mass spectrometer, and design of high-sensitivity instruments with very high mass ranges. It is hoped that comparative studies to identify, quantitate, and characterize proteins differentially expressed in normal versus diseased cells would give insight into mechanisms of pathogenesis and allow the development of a way to control both the etiology and the course of diseases.

Staining methods in gel electrophoresis, including the use of multiple detection methods.

Polyacrylamide gel electrophoresis is a reliable and widely used technique for the separation, identification and characterization of proteins and protein mixtures. With the introduction of high resolution two-dimensional polyacrylamide gel electrophoresis in 1975 upward to 2000 individual polypeptides spots are easily separated on a single electrophoretic gel thereby necessitating the availability of highly sensitive protein detection methods. Although a plethora of protein-staining and -visualization protocols have been described utilizing both radioactive and non-radioactive reagents, many times the use of mono-dimensional detection procedures is insufficient to address the experimental questions asked. The present review highlights the utilization of combined protein-labeling and -staining methodologies in gel electrophoresis including selected applications in polyacrylamide gels and solid membrane matrixes.

Influence of conjugation reactions on the mutagenicity of aromatic amines.

2-Acetylaminofluorene (AAF) and 2-aminofluorene (AF), as well as their N-hydroxylated metabolites, N-OH-AAF and N-OH-AF, were studied for mutagenic effects in Salmonella typhimurium with rat- and mouse-liver S9 and microsomal subfractions in the presence of cofactors for glucuronidation and glutathione (GSH) transfer. Addition of UDPGA did not affect the mutagenicity of AAF, AF or N-OH-AAF under any experimental condition. Addition of GSH, on the other hand, markedly inhibited AAF, AF and N-OH-AAF. This seemed to be due to the direct effect of GSH, and not through an enzyme-catalyzed conjugation. Further, GSH inhibited the direct mutagenicity of N-OH-AF.

Guanethidine N-oxidation in human liver microsomes.

The capacity of human liver microsomes to N-oxidize guanethidine from 25 subjects has been assessed. Guanethidine N-oxidation was optimal at pH 8.5 and proceeded at only 16% of the maximal rate at pH 7.4. The mean rates of guanethidine N-oxidation at pH 8.5 and 7.4 were 2.46 +/- 0.89 (mean +/- s.d., n = 25) and 0.38 +/- 0.22 (mean +/- s.d., n = 22), respectively. Interindividual differences in the rate of guanethidine N-oxidation at pH 8.5 and 7.4 were 17- and 11-fold, respectively. The cytochrome P450 inhibitors, proadifen and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), at both pH 8.5 and 7.4 caused less than 20% reduction in the rate of guanethidine N-oxidation by human liver microsomes. These data indicate that guanethidine N-oxidation can be used as a measure of flavin-containing monooxygenase activity in human liver.