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Biomedical subjects

A K Verma

Publications and source records attributed to A K Verma.

At least 109 records · Page 6Linked to original sources

Sinusoidal endothelial hyperplasia in pathogenesis of perilobular fibrosis in rabbit after experimental ligation of common bile duct.

Development of perilobular hepatic fibrosis in rabbits after experimental ligation of the common bile duct was studied by microscopy. Twelve hours after the ligation, the lobular boundaries assumed prominence by appearance of row(s) of congested sinusoids around the distended perilobular canals of Hering. Seven days later, the lobular laminae limitans around such canals of Hering revealed myxomatous ballooning degeneration forming well defined inter-lobular bands (ILBs). On day 15 post-ligation, the residual sinusoidal endothelial-lining cells in the ILBs manifested hyper chromasia and occasional mitotic activity. It was followed, on days 25 and 35, by proliferation (hyperplasia) of the sinusoidal endothelium cells forming the portal tract like fibrotic tissue in the perilobular bands. These findings, showed the course of interlobular bridging necrosis along the bile preductules at the lobular circumferences and subsequently indigenous origin of the fibrotic tissue in the ILBs from the residual cells in the affected parenchyma independent of the portal tracts.

Animals↗

Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin.

The effects of dietary supplementation of flavonol quercetin on both 7,12-dimethylbenz(a)anthracene (DMBA)- and N-nitrosomethylurea-induced mammary cancer in female Sprague-Dawley rats were determined. Quercetin diet was started 1 wk before intragastric instillation of DMBA (65 mg/kg of body weight) or i.v. injection of N-nitrosomethylurea (50 mg/kg of body weight) and was continued during the entire period (20 wk) of the experiment. Dietary quercetin inhibited both the incidence and the number of palpable rat mammary tumors; rats fed on 2% quercetin had 25% less incidence of mammary cancer, while the average number of mammary tumors per rat was reduced by 39% at 20 wk post-DMBA administration compared to animals on a control diet. In a separate experiment, a 5% quercetin diet elicited a greater inhibitory effect on the induction of rat mammary tumors by DMBA than was observed with a 2% quercetin diet. The inhibitory effect of quercetin on mammary tumor incidence in rats on 2% and 5% diets and on tumor multiplicity in animals on a 5% diet was statistically significant (P less than 0.05). In addition, the risk of the development of a palpable tumor (as determined by the nonparametric estimate of the hazard function) in the quercetin-fed group was lower than the group on control diet throughout the course of the experiment. Furthermore, 5% dietary quercetin significantly inhibited (P less than 0.05), although to a lesser extent than observed in DMBA-induced tumor formation, both the incidence and the number of palpable mammary tumors per rat induced by N-nitrosomethylurea. Dietary quercetin did not elicit any detectable sign of toxicity. The gain in body weight in rats on the quercetin diet and the quantity of diet consumed per rat per week were similar to those for rats on the control diet.

9,10-Dimethyl-1,2-benzanthracene↗

Complete primary structure of a human plasma membrane Ca2+ pump.

cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.

Amino Acid Sequence↗

Inhibition of tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced synthesis of epidermal ornithine decarboxylase messenger RNA and diacylglycerol-promoted mouse skin tumor formation by retinoic acid.

Evidence is presented that inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC; EC 4.1.1.17) by retinoic acid may involve inhibition of protein kinase C-mediated synthesis of ODC mRNA. A single application of 10 nmol of TPA to intact mouse skin led to an increase in the steady state levels of epidermal ODC mRNA; a maximal level of ODC mRNA occurred at about 3.5 h after TPA treatment. TPA-induced increase in ODC mRNA preceded the increase in epidermal ODC activity. Application of 17 nmol of retinoic acid 1 h before application of TPA to mouse skin inhibited the induction of both ODC mRNA and ODC activity. Using the DNA-excess filter hybridization technique, we found that TPA-increased steady state levels of ODC mRNA in primary culture of newborn mouse epidermal cells were the result of enhanced accumulation of newly synthesized ODC mRNA. Furthermore, in a pulse-chase experiment, we could not detect any difference in the half-life of ODC mRNA in epidermal cells after TPA or the vehicle dimethyl sulfoxide treatments; the half-life of ODC mRNA was about 7 h in both cases. Exposure of primary cultures of newborn epidermal cells to retinoic acid, in conjunction with TPA, inhibited the synthesis of ODC mRNA and failed to alter the half-life of ODC mRNA. These results implicate the role of transcription activation in TPA-induced ODC gene expression and indicate that retinoic acid may inhibit TPA-induced ODC gene transcription. We also found that protein kinase C may play a role in the mechanism of inhibition by retinoic acid of ODC gene expression. Supporting evidence is the finding that L-alpha-dioctanoylglycerol, an activator of protein kinase C, is a Stage II mouse skin tumor promoter and the application of retinoic acid 1 h before application of L-alpha-dioctanoylglycerol to mouse skin inhibited the induction of ODC activity and ODC mRNA as well as tumor promotion by L-alpha-dioctanoylglycerol. Taken together, one may conclude that the mechanism of inhibition of TPA-induced ODC by retinoic acid may involve the inhibition of protein kinase C-mediated accumulation of newly synthesized ODC mRNA.

Animals↗

The protein kinase C activator L-alpha-dioctanoylglycerol: a potent stage II mouse skin tumor promoter.

Endogenous diacylglycerol, as produced during ligand-stimulated hydrolysis of phosphatidylinositol, is a physiological activator of protein kinase C, a receptor for the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Diacylglycerol mimics many effects of phorbol ester TPA, but it is not known whether diacylglycerol is a mouse skin tumor promoter. The present studies determined the mouse skin tumor-promoting activity of L-alpha-dioctanoylglycerol (DG), a membrane-permeable diacylglycerol derivative. In two independent experiments with female SENCAR mice, DG at a 2 mumol dose, when applied twice weekly to the initiated mouse skin, failed to promote mouse skin tumor formation. Similarly, DG lacked Stage I tumor-promoting activity; twice weekly applications of DG for 2 wk to the initiated mouse skin followed by twice weekly applications of mezerein (3.3 nmol) for as long as 27 wk elicited only a few papillomas per mouse. DG was found to be a potent Stage II mouse skin tumor promoter. In a typical two-stage tumor promotion experiment, SENCAR mice were initiated by application of 20 nmol of DMBA to their shaved backs. Two wk after initiation, 3.3 nmol of TPA were applied twice weekly for 2 wk (Stage I), and then 2 mumol of DG or 3.3 nmol of mezerein were applied to the skin twice weekly for the entire duration of the experiment (Stage II). Stage II tumor promotion with mezerein and DG resulted in 13.33 +/- 0.88 and 11.13 +/- 1.25 papillomas per mouse, respectively, at 19 wk and the carcinoma incidence, 43% and 33%, respectively, at 27 wk of promotion. The tumor-promoting activity of DG was compared with the parent alcohol glycerol, and it was found that glycerol, at a dose as high as 11 mumol, was not a complete, Stage I or Stage II mouse skin tumor promoter. Both TPA and DG, when applied to mouse skin, induced epidermal ornithine decarboxylase activity. Both TPA and DG activate protein kinase C, but the results presented indicate that TPA and DG differ in their tumor-promoting properties. DG, like mezerein, is a Stage II mouse skin tumor promoter.

9,10-Dimethyl-1,2-benzanthracene↗

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography.

Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins.

Animals↗

Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes.

Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.

Amino Acid Sequence↗

Mechanisms involved in ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate, a potent mouse skin tumor promoter and an activator of protein kinase C.

ODC, the first enzyme in mammalian polyamine biosynthesis, is rapidly induced in response to a wide variety of growth stimuli. However, there is no single mechanism which may explain the rapid turnover of ODC activity. ODC activity has been shown to be regulated at the level of synthesis and degradation, and also by post-translational modifications and an interaction with macromolecules. Our results indicate that TPA-induced ODC activity is regulated at the transcriptional level. An initial signal in ODC induction by TPA is not clear. We have suggested that TPA-increased accumulation of epidermal prostaglandins is required, but not sufficient, for ODC induction by TPA. Others have suggested the role of lipoxygenase product(s) in ODC induction. The role of the microtubule-containing system in regulation of ODC induction has been shown. The involvement of cyclic nucleotides in ODC induction by TPA is controversial. Also, generation of free radicals appears to be involved in ODC induction by TPA. Data summarized in this chapter indicate that activation of PKC may be an initial step in ODC induction by TPA.

Animals↗

Involvement of protein kinase C in the transcriptional regulation of ornithine decarboxylase gene expression by 12-O-tetradecanoylphorbol-13-acetate in T24 human bladder carcinoma cells.

We have analyzed the molecular mechanisms involved in ornithine decarboxylase (ODC) induction by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in T24 cells, an easily manipulable human epithelial cell line. The addition of as low as 10(-9)M TPA to T24 cells, cultured in a serum-free medium, resulted in ODC induction, with peak ODC activity occurring at about 6 h after TPA treatment. The induction of ODC activity correlates with the steady-state levels of ODC mRNA increased by TPA in T24 cells. TPA treatment did not elicit any change in the size (2.1 kb) of ODC mRNA as determined by Northern blot analysis by hybridization to nick-translated 32P-labeled cDNA prepared from either mouse or human ODC mRNA. Using the DNA-excess filter hybridization technique, we found that increased steady-state levels of ODC mRNA after TPA treatment may be the result of enhanced accumulation of newly synthesized ODC mRNA. The magnitude of the induction of ODC activity was proportional to the amount of ODC mRNA synthesis caused by TPA. In a pulse-chase experiment, we failed to detect any difference in the half-life of ODC mRNA in T24 cells after treatment with either TPA or the vehicle ethanol; the half-life of ODC mRNA was about 6 h in both cases. Furthermore, as determined by the "nuclear runoff transcription assay," the rate of transcription of ODC-gene was increased by treatment of T24 cells with TPA. These results provide direct evidence of the role of transcription-initiation in ODC-gene expression. The examination of the role of protein kinase C in ODC-gene transcription revealed that TPA or diacylglycerol did not induce the synthesis of ODC mRNA in PKC-deficient T24 cells. Taken together these results indicate that the TPA-increased synthesis of steady-state levels of ODC mRNA in T24 cells may be mediated by protein kinase C and is regulated at the transcriptional level.

Diglycerides↗

Co-localization of erythrocyte Ca++-Mg++ ATPase and vitamin D-dependent 28-kDa-calcium binding protein.

We have previously shown that the human kidney distal convoluted tubule (DCT) contains epitopes of the human erythrocyte Ca++-Mg++ ATPase pump (J Clin Invest 80: 1225-1231, 1987). To determine whether vitamin D-dependent 28-kilodalton-calcium binding protein (28kDa-CaBP)and Ca++-Mg++ ATPase are present in the same cells of the human kidney, kidney tissue was examined for immunoreactivity with antibodies directed against these proteins. Double-label immunohistochemistry showed that a majority of the distal convoluted tubules contain epitopes to both of these proteins. portions of the distal nephron which were positive for 28kDa-CaBP did not show anti-Ca++-Mg++ ATPase antibody binding. All other portions of the nephron were negative for both proteins. Western blot analysis of kidney homogenates by 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), showed binding of an anti-Ca++-Mg ATPase monoclonal antibody to a major band of Mr = 140,000. Western blots of kidney homogenates by 10% SDS-PAGE also showed binding of an anti-28kDa-CaBP polyclonal antibody to a protein band at Mr = 28,000. Incubation of parallel blots from the same 10% gel with 45CaCl2 demonstrated that the Mr = 28,000 band binds calcium. This work demonstrates, for the first time, that epitopes of vitamin D-inducible 28kDa-CaBp and human erythrocyte Ca++-Mg++ ATPase pump are present in the same cells of the human kidney. Previous work in our laboratory has shown that 28kDa-CaBP binds calcium in a manner analogous to calmodulin, a known regulator of the erythrocyte Ca++-Mg ATPase pump (J Biol Chem, 1987).(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

Systemic embolization from thrombus in normal left ventricles.

It is extremely rare for a thrombus to form in a normal functioning left ventricle. We present two patients in whom this occurred without any plausable explanation. In both instances the patients presented with systemic emboli and had no heart disease or hypercoagulable state. The diagnosis was made by two-dimensional echocardiography and subsequently confirmed during surgery.

Adult↗

Inhibition of phorbol ester-induced ornithine decarboxylase gene transcription by retinoic acid: a possible mechanism of antitumor promoting activity of retinoids.

We have shown that retinoic acid, applied either to the skin or administered in diet, inhibits skin tumor promotion by TPA. Retinoic acid does not inhibit the initiation step of mouse skin carcinogenesis. Our results indicate that retinoic acid inhibits both stage I and stage II of tumor promotion, and the inhibition of tumor promotion depends upon the duration of retinoic acid treatment. The inhibition of skin carcinogenesis by retinoic acid is not universal; retinoids exhibit specificity towards carcinogens and tumor promoters. In conclusion, the results presented indicate that the inhibition of TPA-induced ODC gene expression may be one of the mechanisms contributing to the antitumor promoting property of the retinoids. However, other mechanisms concerning the effect of retinoic acid on chromatin structure (Porter et al., 1986), glycoprotein synthesis (Levin et al., 1983), peptide growth factors (Sporn et al., 1986), induction of transglutaminase (Lichti and Yuspa, 1985) and the host-immune system (Dennert, 1985) may also explain the molecular basis of retinoid action.

Animals↗

Inhibition of both stage I and stage II mouse skin tumour promotion by retinoic acid and the dependence of inhibition of tumor promotion on the duration of retinoic acid treatment.

Retinoic acid is a potent inhibitor of mouse skin tumor promotion by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have further evaluated the effect of retinoic acid on the stages of tumor promotion and also analyzed the effect of duration of retinoic acid treatment on mouse skin tumor promotion by TPA. In a number of independent experiments, either with female CD-1 or SENCAR mice, we failed to observe a specificity of inhibition by retinoic acid of either Stage I or Stage II tumor promotion. In a typical experiment with SENCAR mice, application of 34 nmol of retinoic acid concurrently with each application of either TPA (3.2 nmol) or mezerein (3.2 nmol) to initiated (with 10 nmol 7,12-dimethylbenz(a)anthracene) skin equally inhibited promotion of skin papilloma formation. Furthermore, sustained inhibition of tumor promotion by retinoic acid required a continuous application of retinoic acid in conjunction with each promotional treatment with TPA; if retinoic acid treatment was discontinued, TPA treatment elicited tumor formation. These results indicate: (a) retinoic acid inhibits both Stage I and Stage II of tumor promotion; and (b) inhibition of tumor promotion exhibits retinoic acid dependency.

Animals↗

Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis.

Topical treatment of mouse skin with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) results in an array of biochemical alterations, one of the earliest being a more than 200-fold transient induction of epidermal ornithine decarboxylase (ODC) activity. There is an excellent correlation between the induction of epidermal ODC activity and changes in the level of immunoreactive ODC protein following a single TPA treatment to skin. Both ODC activity and protein levels peak at 4.5 h after TPA treatment and rapidly fall to basal levels by 24 h. Cycloheximide treatment of mice in which ODC had been previously induced by TPA indicated a similar rapid turnover of both ODC catalytic activity and protein levels. Northern blot analysis of polyadenylated RNA isolated from mouse epidermis after a single TPA treatment revealed the stimulation of one species of ODC mRNA of 2.0 kilobases with a maximum at 3.5 h declining by 16 h. The same-sized species of ODC mRNA was detected 4.5 h after multiple biweekly treatments with TPA as well as in mouse papillomas and carcinomas not treated with TPA for at least 1 week. Southern blot analysis of EcoRI or BamHI digests of DNA derived from mouse liver, papillomas, or carcinomas revealed no ODC gene amplification or rearrangement during neoplastic progression. These observations indicate that the induction of epidermal ODC activity following TPA treatment results in a transient increase in the steady state levels of ODC mRNA and in the rate of synthesis of ODC protein, in contrast to epidermal tumors where the levels of ODC mRNA and protein are constitutively elevated.

Animals↗

Heteroarotinoids. Synthesis, characterization, and biological activity in terms of an assessment of these systems to inhibit the induction of ornithine decarboxylase activity and to induce terminal differentiation of HL-60 cells.

The synthesis of certain heteroarotinoids has been achieved, namely the systems (2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethyl-6 -thiochromanyl)-2,4,6-heptatrienoic acid (1a), ethyl (2E,4E,6E)-3,7-dimethyl-7- (1,2,3,4-teterahydro-4,4-dimethyl-6-thiochromanyl)-2,4,6- heptatrienoate (1b), (2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethyl-6 -chromanyl)-2,4,6-heptatrienoic acid (1c), 2-phthalimidoethyl 3,7-dimethyl-7-(1,2,3,4-tetrahydro-4 4-dimethyl-6-thiochromanyl)-2,4,6-heptatrienoate (1d), methyl (E)-p-[2-(4,4- dimethyl-6-chromanyl)-1-propenyl]benzoate (2a), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzyl alcohol (2b), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzonitrile (2c), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzaldehyde (2d), methyl 4-[2-(2,3-dihydro-3,3-dimethyl-5-benzofuranyl)-1-propenyl] benzoate (3a), and (E)-p-[2-(2,3-dihydro-3,3-dimethyl-5-benzofuranyl)-1- propenyl]benzoic acid (3b). Characterization via elemental, IR, 1H NMR, and 13C NMR analyses was completed for these heterocycles. The biological activity of these heteroarotinoids was assayed by either the suppression of the 12-O-tetradecanoylphorbol 13-acetate (TPA) induced synthesis of ornithine decarboxylase (ODC) in mouse skin or the induction of differentiation of human (HL-60) promyelocytic cells. In the ODC assay, systems 1a-c exhibited strong activity (within 10% of or less than the control) whereas alcohols 2b and 3a showed good activity (within 50% of the control) as compared to either 13-cis-retinoic acid or trans-retinoic acid. Moderate activity was observed with 2a and 2b while 1d and 2c were essentially inactive. With the HL-60 assay, 1a and 1c were approximately 2- and 5-fold less active, respectively, than trans-retinoic acid. In contrast, 2a, 3a, and 3b induced differentiation of only a very small percentage of the cells. Acids 1a and 1c were the most active heteroarotinoids in the two biological assays. Consequently, the presence of the heteroatom does not eradicate the activity of the heteroarotinoids and thus they may have potential as chemotherapeutic agents.

Cell Differentiation↗

Escherichia coli infection of the urinary bladder: induction of tumours in rats receiving nitrosamine precursors and augmentation of bladder carcinogenesis by N-nitrosobutyl (4-hydroxybutyl)amine.

Experimental introduction of Escherichia coli type 04 into the subserosa of the urinary bladder of female Fischer 344 rats produced chronic bacterial infection in more than 90% of animals. Groups of rats with bacterial infection were given sodium nitrate and either piperazine (Group 1) or dibutylamine (Group 2) in the drinking-water. Control, noninfected animals received nitrate and either piperazine (Group 3) or dibutylamine (Group 4). At 40 weeks, transitional-cell carcinomas of the bladder were detected in 9/30 rats in Group 1 compared to 0/34 in Group 3 (p less than 0.0005), and in 11/34 rats in Group 2 compared to 0/32 in Group 4 (p less than 0.0003). Early changes were examined by scanning and transmission electron microscopy as well as autoradiography. Preneoplastic liver foci were detected in infected groups of animals receiving amine and nitrate, indicating reabsorption of the carcinogen synthesized in situ to induce distant organ transformation. In another experiment, E. coli infection augmented bladder carcinogenesis by N-nitrosobutyl(4-hydroxybutyl)amine (NBHBA), as indicated by earlier appearance of bladder tumours (six weeks compared to nine weeks) and, after 25 weeks, higher incidences of transitional-cell carcinomas (41/46 compared to 39/53, p less than 0.05), squamous metaplasia (43% compared to 9%, p less than 0.0001), glandular metaplasia (26% compared to 13%, p less than 0.05) and muscle invasion (30% compared to 11%, p less than 0.01) in the E. coli-infected group receiving carcinogen compared to the noninfected group receiving carcinogen, respectively. These results indicate that bacterial infection of the urinary bladder may play a major role in bladder carcinogenesis, both by helping in-situ nitrosamine synthesis and by augmenting carcinogenesis by nitrosamines.

Amines↗

Increased mouse epidermal ornithine decarboxylase activity by the tumour promoter 12-O-tetradecanoylphorbol 13-acetate involves increased amounts of both enzyme protein and messenger RNA.

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.

Animals↗

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability.

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.

Animals↗