Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.
Bridged-Ring Compounds ; Imidazoles ; Ornithine ; Ornithine Decarboxylase ; Ornithine Decarboxylase Inhibitors ; Putrescine

BACKGROUND: One of major consideration of any identification (ID) system is the cost. Commercial kits, however, are too expensive. The aim of this study was to evaluate the clinical usefulness of the computerized ID system based on the conventional minimal biochemical tests for the identification of clinical isolates of Enterobacteriaceae. METHODS: During June 1996 to April 1997, Enterobacteriaceae were tested by triple sugar iron, motility, indole, ornithine decarboxylase, and citrate, and 2-4 biochemical tests were tested additionally according to the characteristics of colony on MacConkey agar. We also compared the conventional ID system with API Rapid 32E (ATB system, bioMrieux, France) to determine the accuracy of conventional ID system. RESULTS: Among the 3,652 strains of Enterobacteriaceae, 84.4% were identified by conventional tests. The identification rate of Enterobacteriaceae by conventional tests; K. pneumoniae, E. coli, P. stuartii, M. morganii, and P. mirabilis were more than 80%; K. oxytoca, Enterobacter, and S. marcescens were ranged from 70% to 80%; P. vulgaris, P. rettgeri, and C. freundii were less than 70%. Among the 133 isolates tested simultaneously by two ID systems, each of one strain of M. morganii, E. cloacae, and S. marcescens on conventional minimal biochemical tests were identified as E. coli, E. sakazakii, and S. liquefaciens by commercial kit. CONCLUSIONS: Our computerized ID system based on the minimal biochemical tests was found to offer simple, reliable and economic in the identification of clinical isolates of Enterobacteriaceae. And further studies are needed for the improvement of accuracy and identification rate.
Agar ; Citric Acid ; Cloaca ; Enterobacter ; Enterobacteriaceae* ; Iron ; Mirabilis ; Ornithine Decarboxylase ; Pneumonia

OBJECTIVE: To explore the mechanism of alpha-difluoromethylornithine (DFMO) inhibiting ODC activity in the cortex and hippocampus in rats. METHODS: Forty male rats was randomly divided into ischemal control group and DFMO pretreatment group. DFMO was given intravenously half an hour before global cerebral ischemia, and expression of ODC mRNA was measured by comparative reverse transcription-polymerase chain reaction (RT-PCR) in the cortex and hippocampus in rats after 2, 4, 6 h and 8 h of reperfusion. The variations of the expression of ODC mRNA were studied in the DFMO pretreatment group and the ischemal control group respectively. RESULTS: After 2, 4 and 6 h of reperfusion, the expression of ODC mRNA in the cortex and hippocampus in the pretreatment group was lower than that in the ischemia control group significantly (P <0.05, P <0.01), but not at 8 h reperfusion (P > 0.05). CONCLUSION DFMO suppressed the expression of ODC mRNA after different lengths of reperfusion following 10-minute global cerebral ischemia in rats and it may be one of the ways for DFMO to inhibit ODC activity.
Animals ; Brain Ischemia ; metabolism ; Cerebral Cortex ; metabolism ; Eflornithine ; pharmacology ; Hippocampus ; metabolism ; Male ; Ornithine Decarboxylase ; biosynthesis ; genetics ; Ornithine Decarboxylase Inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism

BACKGROUND: The selection of identification (ID) system of Enterobacteriaceae depends mainly on accuracy of identification system, cost of operation and convenience of testing. Commercial ID kits are easy to use but too expensive. Therefore, we designed a computerized ID system based on 10 tubes which were composed of 14 conventional biochemical tests to identify the Enterobacteriaceae and Vibrionaceae. The purpose of this present study was to assess the clinical usefulness of 10 tube system as an identification system for Enterobacteriaceae in clinical microbiology laboratories. METHODS: During the period of January 1998, 189 Enterobacteriaceae and 2 Aeromonas spp. consecutively isolated from clinical specimens were simultaneously identified by 10 tube system and the API rapid ID 32 E. Fourteen conventional biochemical tests used in 10 tube system were lactose, sucrose, and H2S in Kligler's iron agar media; motility, indole, and ornithine decarboxylase in motility-indole-ornithine decarboxylase agar media; citrate, urease, lysine decarboxylase, phenylalanine deaminase, arginine dihydrolase, arabinose, trehalose, and adonitol. Identification program used in 10 tube system were % ID method and ID score method. RESULTS: Among the 191 isolates, agreement rate of identification between 10 tube system and API rapid ID 32 E were 96.0% to the species level and 99.4% to the genus level. And identification accuracy of 10 tube system was 90.6% to the species level and 93.2% to the genus level. CONCLUSIONS: 10 tube system has been shown to be an accurate, cost-effective alternative to the use of commercial kit systems for identification of Enterobacteriaceae.
Aeromonas ; Agar ; Arabinose ; Arginine ; Citric Acid ; Enterobacteriaceae* ; Iron ; Lactose ; Lysine ; Ornithine Decarboxylase ; Phenylalanine ; Ribitol ; Sucrose ; Trehalose ; Urease ; Vibrionaceae

BACKGROUND: To access the accuracy and clinical usefulness of microplate identification (ID) system for the identification of Enterobacteriaceae, we compared microplate ID system with API 20E(bioMerieux, Etoile, France). METHODS: Ninety-two cultures of Enterobacteriaceae and one isolate of Aeromonas species were simultaneously identified by microplate ID system and the API 20E. Twenty biochemical tests used in microplate ID system were lactose, sucrose, and H2S in Kligler's iron agar media; indole, sucrose, raffinose, arabinose, trehalose, adonitol, dulcitol, sorbitol, cellibiose, methy-red, phenylalanine deaminase, ornithine decarboxylase, lysine decarboxylase, arginine dihydrolase, urease, and citrate in microplate; and oxidase test. The identification was obtained by considering percent likelihood(% ID), modal frequency and ID score method. RESULTS: Among the 92 cultures of Enterobacteriaceae and one isolate of Aeromonas species, agreement rate of identification according to the % ID between microplate ID system and API 20E were 90.3% to the species level and 97.8% to the genus level. CONCLUSIONS: For the identification of clinical Enterobacteriaceae isolates, the microplate ID system compares favorably with API 20E in identification accuracy and have the advantage of costsaving and easy to use.
Aeromonas ; Agar ; Arabinose ; Arginine ; Citric Acid ; Enterobacteriaceae* ; Galactitol ; Iron ; Lactose ; Lysine ; Ornithine Decarboxylase ; Oxidoreductases ; Phenylalanine ; Raffinose ; Ribitol ; Sorbitol ; Sucrose ; Trehalose ; Urease

Kidney ischemia and reperfusion injury (IRI) is associated with a high mortality rate, which is attributed to tubular oxidative and nitrative stresses; however, an effective approach to limit IRI remains elusive. Spermidine, a naturally occurring polyamine, protects yeast cells against aging through the inhibition of oxidative stress and necrosis. In the present study, spermidine supplementation markedly attenuated histological damage and kidney dysfunction during IRI. In addition, exogenous spermidine potently inhibited poly(ADP-ribose) polymerase 1 (PARP1) activation and DNA nitrative/oxidative stress following IRI. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly enhanced DNA nitration, PARP1 activation, and functional damage during IRI. Finally, in ODC knockdown kidneys, PARP1 inhibition attenuated histological and functional damage induced by IRI, but not DNA nitrative stress. In conclusion, these data suggest that spermidine protects kidneys against IRI through blocking DNA nitration and PARP1 activation and this finding provides a novel target for prevention of acute kidney injury including IRI.
Acute Kidney Injury ; Aging ; DNA* ; Ischemia* ; Kidney* ; Mortality ; Necrosis ; Ornithine Decarboxylase ; Oxidative Stress ; Poly(ADP-ribose) Polymerases ; Reperfusion Injury* ; Reperfusion* ; RNA, Small Interfering ; Spermidine* ; Transfection ; Yeasts

UV radiation is known to cause photoaging of the skin and is considered one of the leading cause of developing skin carcinogenesis. Melatonin which has a highly lipophilic molecular structure facilitating penetration of cell membranes and serving as an extra- and intracellular free radical scavenger has been demonstrated to protect photodamage of skin affected by UV exposure. In this study, we have examined the role of melatonin in response to UVB induced photodamaging process, using human skin fibroblasts in vitro. Cell survival curves after UVB irradiation showed dose-dependent decrease. Only 60% of fibroblasts were survived at 140 mJ/cm2 UVB irradiation. By pre-cultivation of cells with melatonin (100 nM), a significant number of cells remained unaffected. After UVB irradiation with 70 mJ/cm2, the level of putrescine was 1.7+/-0.3 fold increased compared to melatonin pre-treated group. In Northern analyses, the transcriptional level of ornithine decarboxylase (ODC) gene expression was increased by UVB irradiation and prohibited by melatonin. These results indicated that melatonin was effectively able to neutralize membrane peroxidation when present in relevant concentration during UVB irradiation and diminishes the UVB-induced increase of polyamine synthesis and ODC gene expression. Collectively, ODC response to UVB induced changes are possibly involves a melatonin or antioxidant sensitive regulatory pathway in normal human skin fibroblast.
Antioxidants/*pharmacology ; Apoptosis/drug effects/radiation effects ; Fibroblasts/*drug effects/*radiation effects ; Human ; Melatonin/*pharmacology ; Ornithine Decarboxylase/biosynthesis/genetics ; Polyamines/*metabolism ; *Ultraviolet Rays

A cirrhosis risk score (CRS) comprised of single nucleotide polymorphisms (SNPs) in seven genes that predicts the risk of cirrhosis in Caucasian hepatitis C has been reported. The present study was to evaluate the association of 11 separate but related SNPs and the CRS with cirrhosis risk in Chinese hepatitis B patients. A total of 563 Chinese subjects with persistent HBV infection (349 with evident liver cirrhosis and 214 without cirrhosis clinically or pathologically) were studied. The candidate SNPs were detected with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. The allele frequency and genotype distribution of each polymorphism as well as the CRS value within the cirrhosis and non-cirrhosis subjects were compared. The rs2679757 polymorphism of the antizyme inhibitor 1 (AZIN1) gene was associated with the risk of cirrhosis (x2 = 6.79, P = 0.03, odds ratio for GG+AG versus AA = 1.63, 95% confidence interval = 1.13-2.35). A gene variant (rs886277) in the transient receptor potential cation channel subfamily M, member 5 gene (TRPM5) was associated with liver cirrhosis, but did not reach statistical significance (x2 = 5.77, P = 0.06). Two SNPs (rs4986791, rs62522600) are not polymorphic in Chinese. Genotype frequencies of other SNPs were not different between the cirrhosis and non-cirrhosis groups. The overall CRS values were not different between the cirrhotic and non-cirrhotic groups (median value 0.57 versus 0.62, Z = -1.05, P = 0.29). SNP rs2679757 in the AZIN1 gene is associated with the risk of HBV-related liver cirrhosis in Chinese. The CRS for Caucasian population has limited applicability for predicting liver cirrhosis in Chinese hepatitis B patients. SNPs associated with cirrhosis prognosis in hepatitis B patients and liver diseases with other etiologies warrant further clinical validation.
Adult ; Carrier Proteins ; genetics ; Female ; Gene Frequency ; Genotype ; Hepatitis B ; genetics ; Humans ; Liver Cirrhosis ; genetics ; Male ; Middle Aged ; Ornithine Decarboxylase Inhibitors ; Polymorphism, Single Nucleotide

OBJECTIVEIn order to explore if power frequency magnetic field (PFMF) can act as cancer promoter or be synergistic with phorbol 12-myristate 13-acetate (TPA) in cancer promotion, the effects of 50 Hz MF on gap junction intercellular communication (GJIC) of astrocytes were observed.

METHODSFluorescence redistribution after photobleaching (FRAP) was adopted to observe the recovery of fluorescence intensity in the bleached cells thus to estimate intercellular communication by gap junction. Comparative fluorescence intensity recovery rate (CFIRR) was as evaluation index. The effects of 50 Hz MF alone or with TPA on GJIC of astrocytes were studied.

RESULTSAfter 3 ng/ml TPA treatment for 1 hour, M(d) of CFIRR was 4.53%/min, whereas that in the control group was 9.74%/min (H = 12.084, P < 0.005). After exposure to 0.8 and 1.6 mT magnetic field for 24 hours respectively, M(d) of CFIRR was 8.25%/min and 6.68%/min respectively, no significant difference from that of control (H = 32.617, P > 0.05). After exposure to 0.8 and 1.6 mT magnetic field for 23 hours then combined with 3 ng/ml TPA treatment for 1 hour, M(d) of CFIRR was 3.32%/min and 2.85%/min respectively, also no significant difference from that in the group treated with 3 ng/ml TPA alone (H = 2.589, P > 0.05).

CONCLUSION50 Hz MF (within 0 - 1.6 mT) alone could not inhibit GJIC of astrocytes; with TPA, could not enhance the inhibition of TPA on GJIC of astrocytes. But with MF intensity increasing, the inhibition of MF on GJIC showed elevated tendency.

Animals ; Astrocytes ; radiation effects ; ultrastructure ; Cell Communication ; radiation effects ; Electromagnetic Fields ; adverse effects ; Gap Junctions ; radiation effects ; Ornithine Decarboxylase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tetradecanoylphorbol Acetate ; pharmacology

Haemophilus paraphrophilus is a normal inhabitant of the naso- and oropharynx and has been rarely reported as a cause of endocarditis. H. paraphrophilus is a slow-growing and fastidious gram-negative bacterium and belongs to the HACEK group. We experienced a case of infective endocarditis due to H. paraphrophilus. The organism was repeatedly isolated from the blood cultures of a 60 year -old patient presenting with high fever, chills, cardiac murmur, and change of mental state. The patient had a history of mitral and tricuspid valve replacements and had been followed up for complications of cirrhosis of liver such as esophageal varix and oral bleeding. The isolate was identified as H. paraphrophilus by the characteristics, including factor V requirement, negative indole, urea and ornithine decarboxylase and acid production from glucose and lactose. Antimicrobial susceptibility testing by the disk diffusion method showed that the organism was susceptible to ampicillin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole, cefotaxime, ceftazidime, aztreonam, imipenem, and ciprofloxacin. The patient expired on hospital day 8, probably due to complications of cirrhosis of liver To our knowledge, this is the first case of infective endocarditis caused by H. paraphrophilus in Korea.
Ampicillin ; Aztreonam ; Cefotaxime ; Ceftazidime ; Chills ; Chloramphenicol ; Ciprofloxacin ; Diffusion ; Endocarditis* ; Esophageal and Gastric Varices ; Factor V ; Fever ; Fibrosis ; Glucose ; Haemophilus paraphrophilus* ; Haemophilus* ; Heart Murmurs ; Hemorrhage ; Humans ; Imipenem ; Korea ; Lactose ; Liver ; Ornithine Decarboxylase ; Oropharynx ; Tetracycline ; Tricuspid Valve ; Urea