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Oesophageal cancer is a significant cause of cancer related mortality, with increasing incidence worldwide. Ornithine decarboxylase (ODC) is an enzyme involved in polyamine synthesis and cellular proliferation, and ODC expression and activity has been implicated as a prognostic marker of oesophageal cancer. This study aimed to develop and optimise an in vitro (13)C-stable isotope assay for ODC activity as a non-invasive marker of oesophageal cancer. Experiments were performed in triplicate (n  =  3/group/cell line) using Caco2, HeLa, Flo-1, OE33, TE7 and OE21 cell lines (colorectal, cervical, oesophageal adenocarcinoma and oesophageal squamous carcinoma respectively). Following addition of 2mM (13)C-ornithine to cells, 10 ml gas samples were collected from the headspace every 20 min for a total of five hours. Gas samples were analysed using isotope ratio mass spectrometry to quantify (13)CO2. Assay specificity was determined using the selective ODC inhibitor, N-(4'-Pyridoxil)-Ornithine(BOC)-OMe (POB). All data is expressed as δ (13)CO2 from baseline. High ODC activity was detected by (13)C-ornithine assay in Caco2 (32.00  ±  1.12 δ (13)CO2) in contrast to HeLa cells (5.44  ±  0.14 δ (13)CO2) cells. POB inhibited activity in Caco2 cells to 12.87  ±  1.10 δ (13)CO2. Differential ODC activity was detected in all oesophageal cancer cells, and 53 h incubation of cell lines with POB reduced activity by 72%, 56%, 64% and 69% in the Flo-1, OE33, OE21 and TE7 cell lines respectively. We have shown that ODC activity can be selectively detected by a non-invasive, stable-isotope (13)C-ornithine assay. ODC activity was detected in all oesophageal cancer cell lines in vitro. Further studies are indicated to quantify ODC activity in oesophageal cancer patients.

Background: Arginine may play important roles in tumor progression by providing ornithine for polyamine biosynthesis, required for cell growth. The aim of this work was to determine the expression of arginine metabolic pathway enzymes in head and neck squamous cell carcinoma (HNSCC) in northeast India., Materials and Methods: The expressions of arginase isoforms (ARG1 and ARG2), ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) were examined in fifty paired HNSCC and adjacent non-tumor tissues by immunohistochemistry. Immunocytochemistry, semiquantitative reverse transcription sq-PCR and quantitative real-time qPCR were used to assess protein and mRNA expressions in peripheral blood of fifty HNSCC patients and hundred controls., Results: ARG1 and ODC protein and mRNA were strongly expressed in peripheral blood from HNSCC patients. No ARG2 expression was observed. In vivo, expression of ARG1, ARG2 and ODC was significantly higher in tumor than in non-tumor tissues. Most tumors expressed low levels of OAT, with no difference in tissues or blood, compared to controls. The absolute extent of maximal ARG1 upregulation with qPCR showed 6.23 fold increase in HNSCC., Conclusions: These findings strongly suggest that in HNSCCs, the ARG1 pathway is stimulated leading to the formation of polyamines as indicated by higher ODC expression, which promote tumor growth.

Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).

Previous studies from this laboratory have dealt with the purification and biochemical characterization of ornithine decarboxylase (ODC) from Entamoeba histolytica. Enzyme compartmentalization has been described as a major mechanism in the regulation of polyamine metabolism. However, the subcellular location of ODC in the human parasite has remained unresolved. To examine this issue, we cloned the full-length gene (Ehodc) encoding for the parasite enzyme, whose open reading frame encodes for a peptide of 412 amino acids with an estimated molecular mass of 46kDa that exhibits similarity to other ODCs. Heterologous overexpression of the gene allowed us to purify the recombinant protein (rEhODC) by metal affinity chromatography. The purified polypeptide was used to raise heteroclonal antibodies that were utilized to localize the enzyme in situ by immunofluorescence and confocal microscopy. EhODC was observed to be associated with the plasma membrane, in vesicles close to the plasma membrane and in the EhkOs organelle.

Ornithine decarboxylase (ODC) and the antizyme inhibitors (AZIN1 and AZIN2), regulatory proteins of polyamine levels, are antizyme-binding proteins. Although it is widely recognized that ODC is mainly a cytosolic enzyme, less is known about the subcellular distribution of AZIN1 and AZIN2. We found that these proteins, which share a high degree of homology in their amino acid sequences, presented differences in their subcellular location in transfected mammalian cells. Whereas ODC was mainly present in the cytosol, and AZIN1 was found predominantly in the nucleus, interestingly, AZIN2 was located in the ER-Golgi intermediate compartment (ERGIC) and in the cis-Golgi network, apparently not related to any known cell-sorting sequence. Our results rather suggest that the N-terminal region may be responsible for this particular location, since its deletion abrogated the incorporation of the mutated AZIN2 to the ERGIC complex and, on the other hand, the substitution of this sequence for the corresponding sequence in ODC, translocated ODC from cytosol to the ERGIC compartment. Furthermore, the coexpression of AZIN2 with any members of the antizyme family induced a shift of AZIN2 from the ERGIC to the cytosol. These findings underline the complexity of the AZs/AZINs regulatory system, supporting early evidence that relates these proteins with additional functions other than regulating polyamine homeostasis., (2009 Wiley-Liss, Inc.)

Nitric oxide (NO) and polyamines are critical for implantation and development of conceptuses (embryo and extraembryonic membranes), but mechanisms regulating their biosynthesis in uteri and conceptuses are largely unknown. This study determined the effects of the estrous cycle, pregnancy, progesterone, and interferon tau (IFNT) on expression of NO synthases (NOS1, NOS2, and NOS3), guanosine triphosphate (GTP) cyclohydrolase (GCH1, the key enzyme in de novo synthesis of tetrahydrobiopterin, a cofactor for NO production), and ornithine decarboxylase (ODC1) in uterine endometria in cyclic ewes (Days 10-16) and pregnant ewes (Days 10-20). The mRNAs and proteins for NOS1 and ODC1 were most abundant in uterine luminal (LE) and superficial glandular (sGE) epithelia, and abundance was affected by day of estrous cycle and early pregnancy. NOS2, GCH1, and NOS3 mRNAs were detected in very low abundance in uterine epithelia and stromal cells in both cyclic and pregnant ewes. NOS1 mRNA also was expressed very weakly in conceptuses, whereas NOS3 mRNA was abundant in the trophectoderm and endoderm of conceptuses, as were total NOS1 and NOS3 proteins, inhibitory p-NOS1 protein, and stimulatory p-NOS3 protein. GCH1 mRNA was abundant in the trophectoderm and endoderm of conceptuses between Days 13 and 15 of pregnancy and then decreased thereafter, whereas ODC1 mRNA abundance increased in conceptuses between Days 13 and 18 of pregnancy. GCH1 protein was localized primarily in the nuclei of trophectoderm and endoderm, and its abundance decreased after Day 14 of pregnancy, whereas ODC1 protein was more abundant in the trophectoderm than in the endoderm between Days 13 and 18 of pregnancy. Progesterone stimulated NOS1 and GCH1 expression in LE/sGE and glandular epithelia, whereas IFNT inhibited NOS1 expression in these cell types. Thus, biosynthesis of NO and polyamines in ovine uterine endometria and conceptuses is potentially regulated at transcriptional, translational, and posttranslational levels to favor conceptus development and implantation.

The paper describes a new procedure for evaluating the severity of childhood celiac disease, the basis of which is to determine the salivary activity of the enzyme ornithine decarboxylase (ODC). The salivary ODC activity index of 0.0001 to 0.0080 ncat/ml suggests severe celiac disease; that of 0.0081-0.0145 and 0.0146-0.0410 ncat/ml indicates its moderate and mild forms, respectively. The development of this procedure is urgent since the typical forms of celiac disease, which make the determination of the degree of the disease difficult in most cases.

Background: Up to 30% of asthmatic subjects are smokers, and smoking might be an important contributor to asthma pathology. Inducible nitric oxide synthase (iNOS), ornithine decarboxylase (ODC), and arginase I are involved in the arginine pathway. We have shown that arginase I and iNOS are upregulated in asthma. Smoking asthmatic subjects are reported to have low exhaled nitric oxide levels. The effect of cigarette smoking on the expression of arginase I in asthma is unknown., Objectives: The aims of this study were to investigate the expression of arginase I, ODC, and iNOS in asthmatic airways of smokers and nonsmokers and in vitro after nicotine stimulation., Methods: Endobronchial biopsies were performed on 24 steroid-naive subjects with mild asthma: 12 smokers and 12 nonsmokers. Arginase I, ODC, and iNOS levels were assessed by means of immunohistochemistry and in situ hybridization (arginase I). In vitro stimulation of airway cells with nicotine was performed, followed by real-time PCR., Results: Arginase I, ODC, and iNOS were expressed in the epithelium and smooth muscle bundles of both subgroups of asthmatic subjects. There was an increase of arginase I and ODC immunoreactivities in smoking compared with nonsmoking asthmatic subjects. There was no significant difference in immunoreactivity for iNOS between groups. Nicotine induced a 2-fold increase in arginase I and ODC expression in airway epithelial cells and fibroblasts., Conclusion: This study demonstrates that the expression of arginase I and ODC is increased in airways of smoking compared with nonsmoking asthmatic subjects and in vitro by nicotine., Clinical Implications: Increased expression of arginase I might lead to low exhaled nitric oxide and chronic obstructive pulmonary disease-like airway remodeling in smoking asthmatic subjects.

The involvement of polyamines (PAs) in the interaction between Pinus sylvestris L. seedlings and an ectomycorrhizal fungus Suillus variegatus (Swatz: Fr.) O. Kunze was studied in an in vitro cultivation system. PA concentrations in seedlings were analysed after 1, 3, and 5 weeks in dual culture with S. variegatus, and changes in PA pools were compared with the growth of the seedlings. Pinus sylvestris arginine decarboxylase (ADC) and S. variegatus ornithine decarboxylase (ODC) mRNA transcripts were localized during the formation of mycorrhizas. During mycorrhiza formation, Suillus variegatus ODC transcripts were found in developing hyphal mantle and Hartig net, and P. sylvestris ADC transcripts in specific root parenchyma cells adjacent to tracheids and in mitotic cells of the root apical meristem. However, no unambiguous difference in ADC transcript localization between inoculated and non-inoculated roots was observed. Regardless of the unchanged distribution of ADC transcripts, inoculation with S. variegatus increased free putrescine, spermidine, and spermine concentrations in roots within the first week in dual culture. The concentration of free and conjugated putrescine and conjugated spermidine also increased in the needles due to the fungus. The fungus-induced lateral root formation and main root elongation were greatest between the first and third week in dual culture, coinciding with retarded accumulation or a decrease of free PAs. These results show that accumulation of PAs in the host plant is one of the first indicators of the establishment of ectomycorrhizal interaction between P. sylvestris and S. variegatus in the in vitro system.

Background: Probiotics seem to possess tumour inhibitory properties, but few studies have investigated their actions on the cell proliferation of normal colonic mucosa. The effects of a probiotic mixture (VSL#3) on polyamine biosynthesis, Ki-67 levels and apoptosis in the normal colon of rats were studied., Materials and Methods: For a 4-week period, 20 rats were fed a VSL#3 solution and 20 rats a saline solution. Samples from the colonic mucosa were collected at the end of treatment. Polyamines were detected by HPLC, ornithine decarboxylase activity by a radiometric technique, and apoptosis and Ki-67 by histochemical and immunohistochemical methods., Results: VSL#3 caused a significant decrease in colonic polyamine levels, ornithine decarboxylase activity and Ki-67 compared to controls. A significant increase in the apoptotic index was also observed., Conclusion: Probiotics could also reduce proliferation rates in a condition not affected by hyperproliferative or neoplastic growth, when the normal control mechanisms are still completely effective.

Objective: This study, carried out on 51 patients with multifocal atrophic gastritis (MAG) and 92 age and sex-matched dyspeptic controls, was designed to examine both exocrine (gastric acid) and endocrine (gastrin) gastric secretion before and after therapeutic intervention including Helicobacter pylori eradication and vitamin C treatment., Methods: Fasting and gastrin-releasing peptide-induced gastric acid secretion, serum levels of gastrin and proinflammatory (IL-1beta, IL-8, TNF-alpha) as well as gastric mucosal gene expression of ornithine decarboxylase (ODC), cyclooxygenase 2 (COX-2) and growth factors (epidermal growth factor and transforming growth factor alpha) were determined before and after the eradication of Helicobacter pylori and therapy with large doses (1 g/d) of vitamin C for 3 months., Results: The H. pylori eradication, assessed by C-urea breath test, and vitamin C therapy failed to reverse the histological atrophy of the gastric mucosa but improved significantly the functional status of the atrophied mucosa, especially its exocrine and endocrine secretory activities, attenuated the expression of premalignant markers such as ODC and COX-2, raised the production of growth factors and diminished the release of proinflammatory cytokines., Conclusions: These results indicate that MAG may be considered as an environmental disease of the gastric mucosa, whose functional status can be improved by the eradication of H. pylori combined with antioxidant therapy with large doses of vitamin C.

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.

Background: The polyamines spermidine, spermine, and putrescine are known to be deeply linked with growth processes, gene expression, and extracellular matrix synthesis. Their cellular content depends primarily on the activity of the enzyme ornithine decarboxylase. High levels of ornithine decarboxylase and polyamines have been found in proliferative, inflammatory, and neoplastic pathologies of the oral cavity and in gingival fluid. Difluoromethylornithine (DFMO) selectively inhibits ornithine decarboxylase, thus depleting polyamine content and preventing cell proliferation and synthesis activity. The aim of this study was to investigate whether DFMO treatment could modify the genes involved in cell proliferation and extracellular matrix turnover., Methods: Fibroblasts derived from non-inflamed gingiva were maintained in Dulbecco's modified Eagle's medium (DMEM) plus alpha-difluoromethylornithine for 4 days. At 0, 24, 48, 72, and 96 hours cell number was assessed, polyamine levels were quantified with high performance liquid chromatography (HPLC) method, and transforming growth factor-beta1 (TGF-beta1), c-myc, matrix metalloproteinases (MMP)-1 and 2, collagen type I (COL-I) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR)., Results: Fibroblasts treated with DFMO significantly decreased cell proliferation, ornithine decarboxylase activity, and putrescine levels at all treatment times, spermidine after 72 and 96 hours, and spermine after 96 hours of culture. Total polyamines decreased (P < or =0.01) at 96 hours after DFMO treatment, while c-myc, TGF-beta1, MMP-1 and 2, COL-I mRNA significantly increased. Conversely, TIMP-1 did not show any significant change. The polyamines trend was not correlated to c-myc, TGF-beta1, MMP-1 and -2, and TIMP-1 mRNA levels. Transforming growth factor-beta1 and c-myc mRNA expression were related and correlated to MMP-1 and 2, COL-I and TIMP-1 mRNA trend after DFMO treatment., Conclusions: Our data show that as the polyamine content decreases, TGF-beta1, c-myc, MMP-1 and -2, and COL-I mRNA levels increase, therefore a negative regulatory role of the polyamines on the mRNA expression could be suggested.

In a previous publication, we showed that a clinical trial of DL-alpha-difluoromethyl ornithine (DFMO), in combination with PCV (procarbazine, CCNU, vincristine) increased survival of patients with anaplastic gliomas (WHO III) but not glioblastoma multiforme (WHO IV). We believe that treatment outcome (survival) is inversely related to tumor ornithine decarboxylase (ODC) levels. To prove this, we needed to develop an assay to quantify ODC levels in formalin-fixed tumor tissues, which would enable a retrospective study of tumor biopsy specimens from the landmark clinical trial. We developed an assay using a specific polyclonal antibody coupled to an Alexa fluorescent dye. Transgenic MHC-ODC mice with differing levels of ODC in heart muscle were used to establish the relationship between mean gray-scale intensity and enzymatic ODC activity. We found a direct relationship between mean gray-scale intensity of the ODC antibody coupled to Alexa 647 dye and enzymatic activity. Preliminary analysis of a human glioma tissue array shows that tumor-specific variations in levels of ODC can be semiquantitated. We show that mean gray-scale intensity of astrocytoma:glioblastoma is 1:6 and of anaplastic astrocytoma:glioblastoma is 1:4. We also compared the intensity of antibody to Ki67 coupled with phycoerythrin simultaneously in cells but failed to see a relationship that crossed histologies. We conclude that we can measure levels of ODC in formalin-fixed tumor tissue using an antibody to ODC coupled to Alexa 647 dye, and this will enable us to conduct a future study to correlate survival of patients with gliomas of different histologies treated with DFMO to tumor ODC levels.

A new Vibrio species, Vibrio ponticus, is proposed to accommodate four marine bacteria isolated from sea water, mussels and diseased sea bream (Sparus aurata), at the Mediterranean coast of Spain. Strains are Gram negative, slightly halophilic bacteria that require Na+ ion for growth, oxidase and catalase positive, negative for arginine dihydrolase and ornithine decarboxylase but positive for lysine decarboxylase and indole, and utilize beta-hydroxybutyrate as a sole carbon source. Phylogenetic analysis locate these marine bacteria in the vicinity of the V. fluvialis-V. furnissii clade, sharing with these two species 16S rDNA sequence similarities slightly above 97% (97.1 and 97.3%, respectively). DNA-DNA hybridisation values confirm that the four strains form a genospecies and represent a new species in the genus Vibrio. We propose strain 369T (CECT 5869T, DSM 16217T) as the type strain.

Increased ornithine decarboxylase (ODC) activity, measured biochemically in breast cancers, has been associated with increased risk for recurrence of disease and death. Recently an immunohistochemical (IHC) method for ODC determinations in formalin-fixed paraffin-embedded tissues has been developed. We used this IHC ODC assay to evaluate primary breast cancers from 433 Vietnamese premenopausal women participating in a clinical trial of adjuvant combined hormonal therapy. Using an H SCORE system (intensity of staining 0-3 x percentage of all cells; possible range 0-300), 52% of tumors had an ODC score of < or = 35; 12% had a score of > or = 100. No statistically significant correlations of ODC H SCORES and usual prognostic factors were found; a negative weak correlation with weight was demonstrated (Spearman -0.12; p = 0.01). Using two cutoff scores, high and low ODC groups were similar in prognostic factors, except for high histologic grade which was more common with higher ODC H SCORES. Univariate, Kaplan-Meier and multivariate Cox analyses showed no evidence of relationships of ODC by H SCORE to disease-free or overall survival.

Background and Aims: Locally and systemically acting corticosteroids alter the morphology and transport function of the intestine. This study was undertaken to assess the effect of budesonide, prednisone, and dexamethasone on sugar uptake., Methods: Adult male Sprague Dawley rats underwent transection or resection of 50% of the middle portion of the small intestine, and in vitro uptake of sugars was measured., Results: The 50% enterectomy did not alter jejunal or ileal uptake of glucose or fructose. Prednisone had no effect on the uptake of glucose or fructose in resected animals. In contrast, in resected rats budesonide increased by over 120% the value of the jejunal maximal transport rate for the uptake of glucose, and increased by over 150% ileal uptake of fructose. Protein abundance and mRNA expression of the sodium dependent glucose transporter in brush border membrane (SGLT1), sodium independent fructose transporter in the brush border membrane (GLUT5), sodium independent glucose and fructose transporter in the basolateral and brush border membranes (GLUT2), and Na(+)/K(+) ATPase alpha1 and beta1 did not explain the enhancing effect of budesonide on glucose or fructose uptake. Budesonide, prednisone, and dexamethasone reduced jejunal expression of the early response gene c-jun. In resected animals, expression of the mRNA of ornithine decarboxylase (ODC) in the jejunum was reduced, and corticosteroids reduced jejunal expression of the mRNA of proglucagon., Conclusions: These data suggest that the influence of corticosteroids on sugar uptake in resected animals may be achieved by post translational processes involving signalling with c-jun, ODC, and proglucagon, or other as yet unknown signals. It remains to be determined whether budesonide may be useful to stimulate the absorption of sugars following intestinal resection in humans.

The development of reliable methods for the in vitro testing of sensitivity of cancer cells to various drugs has been a longstanding objective in cancer research and treatment Early attempts to develop individualized chemotherapy were based on clonogenic assays. These attempts failed because of low plating efficiencies. Nonclonogenic assays, such as the MMT test or ATP determinations, are based on metabolic activities and do not reflect the ability of cells to proliferate. To detect proliferation, we selected a universal marker--ornithine decarboxylase (ODC), which is expressed early in the cell cycle and has a short half-life. This marker was detected in hematological cancer cells by quantitative immunohistochemical analyses using an ODC antibody and a FITC-linked second antibody. Drug resistance was detected in five patients, who subsequently died. Lymphocytes from normal individuals were sensitive to all drugs tested, whereas 33 leukemia and lymphoma patients showed different sensitivities to certain drugs. The method also permitted testing of the effect of new drugs on the proliferation of lymphocytes from hematological cancer patients. This test is sensitive, and 100-1,000 cells are required per assay, which can be completed within 2 days. It is very likely that the assay could also be used to test solid tumor patients.

Ovarian development, oviposition, larval eclosion, ornithine decarboxylase (ODC) activity, ovarian, testis and ejaculatory apodeme measurements (length, width, and area), and the number of spermatozoa of Anastrepha fraterculus (Wiedemann) were analyzed at alternating (20 degrees/6 degrees C and 20 degrees/13 degrees C) and constant (6 degrees C; 25 degrees C) temperatures. Life span and life expectancy were also analyzed for both genders. All the results suggest that temperature, especially alternating temperatures, increase not only male and female reproductive potential but also their life span and life expectancy. These changes can be a powerful strategy triggered by A. fraterculus as a means to survive the stressful temperature conditions found in winter in the apple production region in Brazil, enabling this species to increase its population density and cause apple damage when spring begins.

Background and Aims: Cellular proliferation and differentiation are regulated by polyamines and their rate-limiting enzyme ornithine decarboxylase (ODC), both of which are correlated with tumor growth, but their role in differentiation is less clear. We investigated the correlation of ODC activity and polyamine levels with tumor stage and grade with respect to sample recruitment., Patients and Methods: We determined ODC activity ([(14)C]CO(2) release), polyamines (HPLC), and histological staging and grading (TNM classification) of tissue samples from 64 patients with colorectal adenocarcinomas., Results: We found the concentrations of putrescine, spermidine, and N(1)-acetyl-spermidine and the ODC activity in tumor tissue to be twice as high as in adjacent normal mucosa. A critical parameter affecting ODC activity was ischemic time, which significantly reduced ODC activity levels in tumors (threefold) and in the surrounding normal tissue (ninefold) when the ischemic period exceeded 1 h. By contrast, polyamine content was not affected by ischemia. Total polyamine and spermine concentrations were higher in T3 and T4 than in T2 tumors, but putrescine was higher in T4 than in T3 and T2 tumors. There were significantly higher levels of total polyamines and spermine in moderately differentiated (G2) than in poorly differentiated (G3) tumors., Conclusion: The lower spermidine/spermine ratio in G2 (0.44) compared with that in G3 (0.64) tumors suggests the involvement of the polyamines in colonic cell differentiation. Polyamine content is thus correlated with the tumor stage.

Oxidative stress plays an important role in hepatocarcinogenesis. Although Sho-saiko-to (TJ-9), a Japanese herbal medicine which has been recently administered to patients with chronic liver disease in Japan, prevents hepatocarcinogenesis, the mechanism by which TJ-9 protects against cancer development is not fully understood. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a DNA adduct by reactive oxygen species, is known as a parameter of genetic risk for hepatocarcinogenesis. To clarify whether the preventive effect on hepatocarcinogenesis by TJ-9 is dependent on 8-OHdG, the effect on 8-OHdG levels by TJ-9 was examined by using high-performance liquid chromatography-mass spectrometry (LC-MS) in a diethylnitrosamine (DEN)-induced hepatocarcinogenesis model of male Fisher rats. TJ-9 reduced the number of preneoplastic cells, detected as the glutathione S transferase P (GST-P)-positive hepatocytes, and inhibited the development of liver tumors. TJ-9 also significantly decreased the formation of 8-OHdG, as indicated by LC-MS and immunohistochemical analysis. In addition, ornithine decarboxylase (ODC) activity and the number of proliferating cell nuclear antigen (PCNA)-positive cells were not altered. An electron paramagnetic resonance spin-trapping technique showed that TJ-9 scavenges hydroxyl radicals in a dose-dependent manner. In conclusion, the results of the present study suggest that TJ-9 prevents hepatocarcinogenesis in association with inhibition of 8-OHdG formation.

Leukocytes ornithine decarboxylase (ODC) activity was measured in normal individuals and in patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP) as well as in accelerated phase (CML-AP), with an aim to examine the role of ODC activity in prognostic evaluation of CML patients. Our results showed that ODC activity was significantly higher in CML-CP (41.02+/-25.57nmol/h per 10(7) cells, P<0.005) and CML-AP (67.71+/-44.42nmol/h per 10(7) cells, P<0.001) patients than in normal subjects (3.12+/-1.34nmol/h per 10(7) cells). Furthermore, patients with CML-AP showed higher ODC activity than CML-CP patients (P<0.005). Patients with CML-CP who converted to accelerated phase within 24 months had higher ODC activity (84.58+/-12.81nmol/h per 10(7) cells) than patients who did not convert to accelerated phase (31.13+/-18.24nmol/h per 10(7) cells). The high value of ODC activity was also associated with less clinico-hematological response. We suggest that ODC activity reflects the neoplastic proliferative activity in CML patients and may serve as an additional prognostic marker.

Among black tea polyphenols, theaflavins were generally considered to be the most effective in cancer chemoprevention. In this study, we examined the inhibitory effects of black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate and theaflavin-3'-gallate, theaflavin-3,3'-digallate (TF-3), and the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema and ornithine decarboxylase (ODC) activity. Topical application of these polyphenols onto the mouse resulted in inhibition of TPA-induced ear edema and skin epidermal ODC activity. The inhibitory order was as follows: TF-3 > TF-2 approximately equal to EGCG > TF-1. Western and Northern blots indicated that TF-3 significantly reduced the protein and mRNA levels of ODC in TPA-treated mouse skin and NIH 3T3 cells, whereas EGCG showed less activity. EGCG and TF-3 were able to inhibit the ODC enzyme activity in vitro. Furthermore, TF-3 also significantly reduced the basal promoter activity of the ODC gene in NIH 3T3 cells that were transiently transfected with ODC reporter plasmid. These results suggested that TF-3 was a potential inhibitor of ODC activity and TPA-induced edema and might be effective in cancer chemoprevention.

The role of circulating tumor markers in providing prognostic information has not been widely studied. In the current study, serum levels of the carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were determined preoperatively in 364 breast cancer patients with no clinical signs of metastasis. The prognostic relevance of these markers for recurrence (175/364) and death of disease (104/175) was determined by Cox multivariate analysis, including the comparison with classical prognostic factors. High levels of both tumor markers were associated with aneuploid tumors with high S-phase fraction and high ornithine decarboxylase activity. CA 15-3 was highly associated with the number of positive lymph nodes and peritumoral lymphatic or blood vessel invasion. No significant associations were found between CEA or CA 15-3 levels and histologic grade, necrosis and steroid receptor status. In univariate analysis, preoperative values, using optimum cutoff values of CA 15-3 (40 U/ml) and CEA (6 ng/ml), were statistically significant for relapse-free survival and overall survival. In multivariate analysis, only node status, DNA ploidy and ornithine decarboxylase activity were independent predictors for relapse-free survival; the estrogen receptor status was a predictor of overall survival. In node-negative patients, ornithine decarboxylase activity was the only factor selected for relapse-free survival. In node-positive patients, the number of lymph nodes and DNA ploidy were the only variables selected for relapse-free survival or overall survival. Estrogen receptor and ornithine decarboxylase activity were excluded for relapse-free survival, but were significant prognostic factors for overall survival., (Copyright 2001 S. Karger AG, Basel)

Individuals who receive life-saving organ transplants and the required immunosuppression often develop secondary cancers. One of the most common secondary cancers is nonmelanoma skin cancer in sun-exposed areas. Attempts to prevent these cancers have not been successful. Difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase (ODC), is a known experimental cancer prevention agent that is being evaluated in a number of human cancer prevention trials. This report describes a Phase I trial in 18 organ transplant recipients, randomized to 1.0 and 0.5 g of DFMO or a placebo, designed to look at short-term toxicities over 28 days as well as the impact of DFMO on two biological parameters, skin polyamines and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC activity. Blood levels of DFMO were also measured. The results indicate that DFMO was well tolerated over the 28-day period. The TPA-induced ODC activity in 3-mm skin biopsies was significantly lowered by 80 and 67% at the two dose levels. Polyamine levels were not affected significantly except for putrescine at the 0.5-g level. Blood levels of DFMO were about two times higher than expected, based on our prior pharmacokinetic studies. Our studies indicate that DFMO is a reasonable agent that should be tested further in larger Phase 2b trials in this population as a chemopreventive agent. TPA-induced ODC activity appears to be a relevant intermediate biological assay.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

Increased ornithine decarboxylase (ODC) activity in human breast cancer specimens has recently been shown to be an independent adverse prognostic factor for recurrence and death. Biochemical measurement of ODC, however, is not practical for routine clinical use. Furthermore, it does not take into account the heterogeneous composition of human breast cancers which contain variable proportions of epithelial and stromal elements. Therefore, we developed an immunohistochemical method for ODC determination which can be applied to formalin-fixed, paraffin-embedded tissue sections. We report here our results in a series of 30 human breast cancer samples. ODC expression was detected most consistently in the malignant epithelial component of the tumors. Twenty-seven of 30 samples stained positive with intensities ranging from 1+ to 3+. The fraction of malignant epithelial cells expressing ODC varied among specimens between 10% and > 90%. When quantitated by H-SCORE, ODC expression was significantly higher in the malignant epithelial component than in normal appearing epithelial cells and stroma admixed within the tumor. Normal mammary tissue adjacent to the cancer was available for analysis in six cases. ODC expression was absent in two (while both cancers were positive) but present in four to a degree which was overall comparable to that observed in the corresponding tumors. We believe that this technique will be useful for future studies aimed at expanding our knowledge of the role of ODC and polyamines (PA) in breast cancer biology.

Barrett esophagus is a premalignant condition that may progress to adenocarcinoma. The risk of developing cancer has been estimated to be approximately 1 in 250 patient-years of observation; however, there appear to be subsets of patients at much higher risk. Risk stratification has previously been determined by histological identification of dysplasia. Several new biomarkers are being tested to help clinicians better determine the risk of cancer development. Although none of these biomarkers has been proven in a prospective study to predict the onset of cancer, they have been correlated with cancer development. Most of these are factors that have been associated with cancer development in other organs. These include assessment of cell proliferation, expression of cyclooxygenase 2, growth factors and oncogenes, secretory factors, cell cycle proteins, adhesion molecules, and aneuploidy and other genetic abnormalities. In addition to their role as potential cancer biomarkers, these factors have increasingly been reported as surrogate markers to monitor the effectiveness of conservative treatments for Barrett esophagus. In this article, biological markers are reviewed for their relevance in Barrett esophagus. Although most biological markers need to be evaluated further and, for most, prospective follow-up studies are lacking, at present abnormal ploidy status, P16 and P53 gene abnormalities, or allelic losses are the most extensively documented.

Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (+/-0.3 degrees C) during the initial 30 min of loose-tube restraint in conditioned animals. When conditioned/tube-trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2-3 min, ACTH increased to 83.2 +/- 31.0 pg/dl, compared to 28.1 +/- 7.7 pg/dl for cage controls, reaching a maximum at 15-30 min (254.6 +/- 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 +/- 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3-10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 +/- 54.8 compared to 31.2 +/- 10.2 pg/dl; corticosterone, 12.2 +/- 6.2 microg/dl compared to 37.1 +/- 6.4 microg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10-20 fold higher (541.8 compared to 27.2-59.1 pg/dl for ACTH) and 2-5 fold higher (45.7 compared to 8.4- 20.0 microg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF-field exposure when the stress-mediated effects were complete further support our conclusion.

Background: Ultraviolet A (UVA) is known to induce the expression of many stress responsive genes due to the generation of reactive oxygen species (ROS). However, UVA's role in inducing metallothionein (MT) gene expression has not been studied. Furthermore, our group demonstrated that UVA enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of ornithine decarboxylase (ODC) activity in mouse skin (1)., Methods: We examined the interaction of UVA, TPA and antioxidants on the induction of MT and ODC mRNA in mouse skin. Female CD-1 mice were exposed to UVA (19 J/cm2) and total RNA was isolated from the skin. Northern blot analysis for MT and ODC mRNAs was performed. ODC activity in mouse epidermis was also determined in some experiments., Results: UVA induced MT mRNA in mouse skin; however, it did not increase ODC mRNA. 1,4-Diazabicylo-[2,2,2]-octane (DABCO), a singlet oxygen scavenger, reduced UVA-mediated induction of MT mRNA by 40%. The data suggest that ROS produced by UVA exposure may contribute to its ability to induce MT mRNA. UVA slightly enhanced TPA-mediated ODC mRNA induction, while it enhanced ODC enzyme activity 70%. UVA additively intensified TPA-mediated MT mRNA induction. alpha-Tocopherol pretreatment inhibited the induction of ODC enzyme activity by TPA treatment combined with UVA exposure (TPA + UVA); however, alpha-tocopherol had less of an inhibitory effect on ODC mRNA induction by TPA + UVA. Curcumin, a plant pigment, dramatically inhibited both TPA- and TPA + UVA-induced expression of ODC and MT genes., Conclusions: These results demonstrate that UVA can induce MT gene expression and enhance TPA-induced ODC and MT gene expression. The data further suggest that these effects are partially mediated by ROS.

Background: Ornithine decarboxylase (ODC) plays an important role in the biosynthesis of polyamines. Induction of ODC and polyamine synthesis has been demonstrated in neoplastic tumors and is thought to be related to the degree of malignancy., Methods: In this study, we investigated a series of basal cell epitheliomas (BCE), Bowen's disease, squamous cell carcinomas (SCC), and metastatic tumors of the skin using an antibody against ODC for immunohistochemistry., Results: Eight of 12 cases of BCE failed to show a positive reaction for ODC. In Bowen's disease, 5 of 13 cases diffusely showed positive reaction for ODC. Fourteen of 15 cases of SCC showed ODC expression, the intensity of which was decreased in the peripheral layer. At higher magnification, the distribution of ODC in the positive SCC cases showed granular and heterogenous patterns. Ten of 14 cases of metastatic skin tumors exhibited positive reactions, and well-differentiated adenocarcinomas tended to show more strongly positive than poorly-differentiated ones., Conclusions: These results support the conclusion that the intensity and the incidence of positive immunohistochemical staining for ODC correlate with the degree of cellular differentiation, and furthermore, that heterogenous distribution of ODC staining may be associated with heterogeneity of tumor cells.

The aim of the present study was to investigate whether temporal changes in polyamine concentration and synthesis could be found in the luminal content and wall tissue of the rumen and abomasum, two organs which have entirely different growth patterns during the first month of life. In the abomasal mucosa there was a marked gradual decrease in the ornithine decarboxylase (ODC) activity during the first month of life, while the ODC activity in the ruminal mucosa was low during the whole experimental period. However, injury of the rumen wall was followed by increased ODC activity. The ODC activity in duodenal mucosa was about 10 times higher than in the ileal mucosa and the ruminal epithelium. In ruminal liquid a clear peak in ODC activity was observed during the period 51-70 days after birth. The polyamine concentration did not parallel the ODC activity, in either the ruminal epithelium or the ruminal liquid. Of the polyamines, the spermine concentration was always highest, and with the exception of duodenal mucosa, the putrescine concentration was lowest. In liver a clear decrease in spermidine concentration from day 1 to about day 60 after birth was observed. Otherwise no marked temporal changes in tissue polyamine concentrations were observed. Two and a half hours after oral administration of 14C-labelled spermine, nearly all of the radioactivity was found in the lumen of the gastrointestinal tract. On the other hand, 1 h after intravenous injection of polyamines the walls of the gastrointestinal tract were strongly labelled. In conclusion, the polyamines needed for ruminal epithelial development seem to come from sources other than the ruminal epithelium itself or the ruminal lumen.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395-1404, 1999)

Enteroinvasive Escherichia coli (EIEC) share important features with Shigella spp., but EIEC strains are difficult to identify because their biochemical reactions are variable, and Sereny tests or other biological and molecular assays are expensive or hard to perform. The aim of this work was to detect probable enteroinvasive E. coli strains by using four biochemical tests, in children under 5 years of age with and without acute diarrhea. 330 strains of E. coli isolated from children with diarrhea, and 660 strains from children without diarrhea were studied. All strains were tested with the following tests: mucus , lysine and ornithine decarboxylase and motility. The strains which were negative to the four tests were tested by Sereny assay. Twelve strains (3.6%) isolated from children with diarrhea were negative to the tests proposed; eleven were lactose positive and only one was lactose negative. Three strains (0.5%) from children without diarrhea were negative to the tests proposed and were lactose positive. All the 15 strains (100%) were positive in Sereny assay. We recommend the use of these four biochemical tests for initial detection of EIEC strains, because their cost is very low and it is feasible carry out them in small diagnostic laboratories.

A new method for in vitro chemosensitivity testing of human lymphoma and leukemia patients has been developed. The method is based on the use of ornithine decarboxylase (ODC), a universal marker of proliferation, which is expressed early during the cell cycle and has a short half-life. This marker was detected by a quantitative immunohistochemical analysis, using an ODC antibody and a FITC-linked second antibody. The in vitro chemosensitivity of lymphocytes from four normal individuals was tested by the immunohistochemical method. Lymphocytes from 25 cancer patients were also examined. In drug-sensitive cells, the intensity of the marker declined in the presence of the drug, whereas resistance to the drug was demonstrated by the presence of the marker. A good correlation was found between the predicted chemosensitivity and the outcome of the therapy. It has been suggested that this approach could be used for in vitro chemosensitivity testing of hematological cancers and most likely also for other malignancies.

In this study, we show that the toxicity of ferric nitrilotriacetate (Fe-NTA) can be correlated with the tissue accumulation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts. It is observed that the toxic manifestations of Fe-NTA gradually increase with the increasing age of animals. A dose of Fe-NTA which produces almost 100% mortality in aged rats causes 70% mortality in adults, 30% in pups, 20% in litters, and less than 10% in neonates. The age-dependent increase in its toxicity is also evident from the data of renal microsomal lipid peroxidation and hydrogen peroxide generation. No significant difference in the generation of H2O2 and induction of renal microsomal lipid peroxidation between saline- and Fe-NTA-treated neonates, litters, and pups could be observed. However, in adult rats, a significant increase in both of the parameters was observed which was even greater in aged rats. On the contrary, renal glutathione levels in these animals show an inverse relationship with the oxidant generation. In neonates, litters, and pups the maximum decrease of glutathione was up to 22%, whereas in adult and aged rats, the depletion was more than 60% of their respective saline-treated controls. Parallel to this data, blood urea nitrogen and creatinine, the indicators of renal damage, show a significant increase in Fe-NTA-treated adult and aged rats only, whereas no significant alterations were observed in other groups. Similarly, the magnitude of ODC induction and [3H]thymidine incorporation was much higher in aged and adult rats in comparison to other groups of animals after Fe-NTA treatment. Additionally, the immunohistochemical localization studies show a significant increase in HNE-modified protein adducts in kidney of adult and aged rats, whereas no significant staining was observed in other groups. A similar increase in the level of protein carbonyls has also been observed with the increasing age of rats. These data suggest that the toxicity of Fe-NTA increases with the increasing age of rats and correlates with the accumulation of HNE-modified protein adducts. It may also be speculated that Fe-NTA-mediated renal toxicity leading to carcinogenesis may be related to the tissue accumulation of HNE-modified protein adducts. However, further studies are needed to establish a definite role of HNE-modified proteins in Fe-NTA-mediated carcinogenesis., (Copyright 1999 Academic Press.)

The effects of the 77 kDa Ju-myo protein, isolated from Drosophila melanogaster, on the development of glutathione S-transferase placental form (GST-P) positive foci in the male F344 rat liver were evaluated using a medium-term bioassay system. No modifying potential was evident in terms of the numbers or areas of GST-P positive foci. Ju-myo protein did not exert any influence on cell proliferation, as reflected by ornithine decarboxylase (ODC) or spermidine/spermine N1-acetyltransferase (SAT) activity and BrdU labeling. These results demonstrated that Ju-myo protein is unlikely to have inhibitory or promoting effects on rat liver carcinogenesis.

Effects of long term administration of Histamine H2-receptor Antagonist (H2-RA) in the treatment of peptic ulcer was studied for the influence to the risk of causing precancerous changes in gastric mucosa. Serum gastrin. ODC activities, polyamine and PCNA labeling index were biochemically and immunohistologically observed. Rat's mucosae with experimentally induced peptic ulcer treated with long term H2-RA, showed no morphological changes but at 30 weeks of H2-RA, had significantly higher value in PCNA labeling index and in polyamine (spermidine) quantity compared to control H2-RA alone and ulcer alone groups. These results suggest that long term administration of H2-RA, even after healing of the ulcer, may cause the gastric mucosa to possess a milieu in favour of precancerous changes, due to the increased proliferative activity of the cells.

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis. An increase in putrescine (a natural polyamine) synthesis after central nervous system (CNS) injury appears to be involved in blood-brain barrier dysfunction, development of vasogenic edema and neuronal death. An improved method is described to determine the ODC activity as well as polyamine levels from the same brain tissue. The polyamine results showed no significant differences from data obtained with the conventional assay. The advantages of this method are to: (1) minimize the number of animals needed for the study, and (2) eliminate any internal inconsistencies resulting from use of two independent groups of animals for ODC and polyamine measurements. Using this method, ODC activities and polyamine levels were measured in cortices and hippocampi from global transient ischemia of gerbils and traumatic brain injury (TBI) of rats.

A two-step Phase I study of piroxicam (PXM) and a-difluoromethylornithine (DFMO) alone and in combination was initiated to assess toxicity and the impact of these drugs on several biological markers. In step 1, 12 subjects with a history of skin cancers were assigned to receive PXM 10 mg every day (q.d.) or 10 mg every other day (q.o.d.). The dosage of PXM 10 mg q.o.d. was tolerated. No changes were seen in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) or urinary polyamine levels. Steady-state serum levels of PXM were consistent with the oral dose level. In step 2, 31 subjects with stage 0 or I nonmelanoma skin cancers, stage A or B prostate or colon cancer, or stage I breast cancer or who had a family history of cancer were randomized to receive DFMO 0.5 g/m2, PXM 10 mg q.o.d., or the combination of DFMO and PXM. In addition to the biological markers of TPA-induced ODC activity in skin biopsies and urinary polyamine levels, we measured urinary 11-dehydrothromboxane B2, a specific metabolite of thromboxane A2. Of the 12 subjects on DFMO/PXM, 2 dropped out for non-drug-related reasons. Three developed grade-2 drug-related toxicities. One subject developed dyspnea that resolved and was able to continue on the study for 6 months. One subject who developed diarrhea that resolved after 5 days was also able to restart the drug without a recurrence. A third subject described intermittent episodes of tinnitus starting 4 h after taking PXM that lasted only 5 s and did not progress on treatment. Comparing the 6-month measurements with pretreatment, DFMO/PXM or DFMO significantly reduced TPA-induced ODC levels (Ps, 0.03 and 0.05). Urinary polyamine levels of spermidine decreased slightly with the DFMO/PXM or DFMO alone, whereas putrescine decreased with PXM alone. Levels of 11-dehydrothromboxane B2 were depressed by PXM and PXM/DFMO. The doses of DFMO/PXM determined in step 2 are potential starting dosages for Phase IIa and IIb chemoprevention trials.

The effect of prolonged administration of the norepinephrine-mimicking agent metaraminol, the alpha1-adrenergic agonist phenylephrine and the alpha2-adrenergic agonist clonidine on the incidence of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the ornithine decarboxylase activity of the gastric cancer and the labeling index of the gastric mucosa were investigated in Wistar rats. Rats received s.c. injections of metaraminol, phenylephrine or clonidine every other day after 20 weeks of oral treatment with MNNG. At week 52, administration of metaraminol and phenylephrine at the higher dose significantly increased the incidence of gastric cancers, the ornithine decarboxylase activity of the gastric cancers and the labeling index of the antral epithelial cells. Administration of phenylephrine at the lower dose and clonidine at both doses had no significant effect on the incidence of gastric cancers, the ornithine decarboxylase activity of the gastric cancers or the labeling index of the gastric mucosa. Our results suggest that adrenoreceptor stimulation enhances gastric carcinogenesis and that such enhancement is mediated through alpha1-adrenoceptors without alpha2-adrenoceptor involvement.

Background: Chronic reflux of duodenal contents into the esophagus of rats produces severe esophagitis and exerts a co-carcinogenic effect on the proliferating cells by enhancing the formation of nitrosamine-induced esophageal carcinomas. We investigated the effect of the different components of the duodenal reflux on the epithelial cell proliferation of the lower esophagus., Methods: Sprague-Dawley rats underwent three surgical reflux models (biliopancreatic, pancreatic, and biliary) and a sham operation. Animals were sacrificed at 72 hours, 6 weeks, and 9 weeks after the operation. Histology and cell proliferation, determined by ornithine decarboxylase activity, polyamine (putrescine, spermidine, spermine) levels, and proliferating cell nuclear antigen labeling index of the basal and suprabasal layers, were studied in the distal esophagus., Results: Both biliopancreatic and pancreatic reflux induced severe esophagitis starting on week 6. Suprabasal proliferating cell nuclear antigen labeling index significantly increased throughout the 9 weeks of the study in the biliopancreatic and pancreatic reflux groups, although this increase was earlier in the former group. Ornithine decarboxylase activity and polyamine levels were significantly increased in the biliopancreatic and pancreatic groups on week 6, decreasing on week 9., Conclusions: Increased esophageal cell proliferation after both biliopancreatic and pancreatic reflux into the lower esophagus may therefore be one mechanism by which duodenal-content reflux stimulates esophageal carcinogenesis in experimental animals.

We examined the changes in ornithine decarboxylase (ODC) immunoreactivity in the hydrocephalic cerebral cortex of HTX rats after decompression by shunt operation. The ODC immunoreactivity reached a very low level after the completion of cortical layer formation, and only faint staining was found on postnatal day (Pd) 11. The ODC immunoreactivity re-appeared after the shunt operation when the operation was done in the early days of life: the ODC immunoreactivity was first found on day 2 after shunting and persisted until day 8 after shunting. However, this was not apparent when the operation was not performed until Pd 14. The re-expression of ODC in hydrocephalic brain after shunting appears to cause resumption of the developmental process by relieving neurons from increased hydrostatic pressure. The dependence of ODC re-expression on the timing of the operation indicates that there may be a period of neocortical decompression that is critical for effective compensatory development, so that when delayed, decompression fails to re-activate the ODC-dependent development.

The activity of ornithine decarboxylase (ODC), an enzyme associated with cellular growth and protein synthesis, was examined in breast muscle, liver, and intestinal tissues of turkeys during a short-term period of feed withdrawal (FW) and following refeeding. Turkeys from a randombred control line were reared under standard management practices to 3 wk of age in battery brooders. Feed was then withdrawn from FW birds for a 48-h period, after which feed was consumed ad libitum. Control birds consumed feed ad libitum throughout the test period. Tissues were collected from 12 birds per treatment following 24 and 48 h of FW and at 6, 12, 24, and 48 h following refeeding for later determination of tissue ODC activity. Activity of ODC was greater in tissue from the small intestine than in liver tissue and both had greater activity than that exhibited by breast muscle. Short-term FW and refeeding produced differential responses in ODC activity of the three tissues examined. Feed withdrawal resulted in a reduction of ODC activity in intestinal tissue, whereas activity was unaffected for liver or breast muscle tissues. Compensatory increases in ODC activity were observed in liver and intestinal tissues; however, the increase was both more rapid and transitory in small intestine than in liver tissue. The ODC activity in breast muscle was largely unaffected by short-term FW and refeeding. Patterns of ODC activity in liver during FW and refeeding closely resembled patterns observed for absolute and relative liver weight. Thus, the results of the present experiment demonstrate that short-term FW and refeeding influence underlying growth mechanisms of supply organs, such as hepatic and intestinal tissue, in addition to affecting overall growth and muscle development.