Evaluation of the Enterotube System for Identification of Members of the Family Enterobacteriaceae

The Enterotube system was evaluated, in parallel with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae, by using bacterial strains from a variety of clinical specimens and from stock cultures. Excellent agreement between the two test systems was obtained with the following reactions: hydrogen sulfide, indole, Simmons' citrate, glucose, and lactose. Agreement was not as good (<85%) with the urea, phenylalanine deaminase, and dulcitol reactions. The Enterotube lysine decarboxylase test was unsatisfactory. The Enterotube method will correctly identify strains of the family Enterobacteriaceae approximately 50% of the time; if identification only as Klebsiella-Enterobacter-Serratia group is needed, the method will be correct 85% of the time. On the basis of this evaluation, the Enterotube system appears to be both simple and rapid for the presumptive identification of these bacteria. Because of the limited usefulness of the lysine decarboxylase test, the results obtained by this test system are less reliable than those obtained by conventional methods.     

Medium and temperature dependence of decarboxylase reactions inAeromonas spp.

Decarboxylase/dihydrolase activities inAeromonas spp. are important as diagnostic tools and indicators of enterotoxin production. We have analyzed the following media at 25°C, 29°C, and 37°C, respectively, for their ability to detect such activities: Møller's, Falkow's, and Fay and Barry's (F&B) containing ornithine, lysine, and arginine, respectively, as well as motility-indole-ornithine (MIO) medium and lysine decarboxylase broth with 0.1% agar (LDC). In order to retain ornithine negativity, but to get as much positivity as possible for arginine, optimal incubation conditions were 29°C for 96 h (Møller), 48 h (Falkow, MIO, and LDC), and 24 h (F&B). The F&B medium proved to be the most sensitive for the detection of lysine decarboxylase, a positive test being highly correlated with the two speciesA. hydrophila andA. sobria, and we suggest its use for routine detection of decarboxylase/dihydrolase activities.In memory of Dr. Sally Jo Rubin.     

The capacity of Enterobacteriaceae species to produce biogenic amines in cheese

The amino acid decarboxylating activity and production of biogenic amines by 104 cheese-associated Enterobacteriaceae species (58 Enterobacter, 18 Serratia, eight Escherichia, seven Hafnia, six Arizona, four Citrobacter and three Klebsiella) were investigated. All strains could decarboxylate at least two amino acids in M?ller's broth and in Niven's medium, and the decarboxylase activity was strain specific. In a laboratory medium containing all free amino acids, all strains could produce more than 100 ppm cadaverine, putrescine was produced by 96% of strains. Tyramine and histamine were produced in the lowest concentrations. A positive correlation existed between cadaverine concentration and Enterobacteriaceae counts in cheese, that may have caused the increase in decarboxylase content. This study suggests that it is possible to limit the presence of cadaverine in cheese, thereby controlling the Enterobacteriaceae counts, a sign of contamination during cheese making and/or storage.     

Ornithine decarboxylase activity in Helianthus tuberosus

Ornithine decarboxylase (ODC, EC 4.1.1.17) was studied in crude extracts of parenchyma slices of dormant tubers activated for 12 h, tuber shoots and shoot apices. It was highest in shoot apices. The enzyme activity was measured by the production of ¹⁴CO₂ from labelled ornithine; V_max was 450 nmol (mg protein)^-1h^-1, K_m for ornithine and pyridoxal phosphate were, respectively, 30 m M and 5μ M . Only when partially purified, the ¹⁴CO₂ production was inhibited by α-difluoromethylornithine, while in crude extracts dithiothreitol was inhibitory. Ornithine and arginine decarboxylase (ADC, EC 4.1.1.19) activities from parenchyma tubers were not greatly altered by exogenously supplemented ornithine, even though its endogenous pool increased. Exogenously supplemented arginine enhanced ornithine decarboxylase activity, whereas putrescine decreased it slightly. The possibility of artifactual activities in the crude extract is also discussed.     

Subcellular Localization of Oxaloacetate Decarboxylase and Its Isolation from Maize Leaves

The activity of oxaloacetate decarboxylase was revealed in leaves of a C₄ plant, maize (Zea mays L.). This activity was unrelated to decarboxylase activities of other enzymes, e.g., NAD-malate dehydrogenase (EC 1.1.1.38) or NADP-malate dehydrogenase (EC 1.1.1.40), and was located in chloroplasts (83.1%). Using a four-step purification procedure, an electrophoretically pure enzyme preparation of oxaloacetate decarboxylase was obtained from maize leaves. The specific activity of the enzyme was 3.150 EU/mg protein, the factor of purification was 40.4, and the yield was 11.0%. The enzyme exhibited Michaelis–Menten kinetics with K _m for oxaloacetate 30 ± 5 M and pH optimum 7.1 ± 0.5. The metabolite-mediated regulation of oxaloacetate decarboxylase activity has been investigated. It is found that sodium chloride (1.0 mM) activates the enzyme, whereas ATP inhibits the enzyme activity.     

Lysine Decarboxylase Activity in Broth and Agar Media

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.     

Evaluation of sulphonamide derivatives acting as inhibitors of human carbonic anhydrase isoforms I,II and Mycobacterium tuberculosis β-class enzyme Rv3273

A series of novel sulphonamide derivatives was obtained from sulphanilamide which was N4-alkylated with ethyl bromoacetate followed by reaction with hydrazine hydrate. The hydrazide obtained was further reacted with various aromatic aldehydes. The novel sulphonamides were characterised by infrared, mass spectrometry, ¹H- and ¹³C-NMR and purity was determined by high-performance liquid chromatography (HPLC). Human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II and Mycobacterium tuberculosis β-CA encoded by the gene Rv3273 (mtCA 3) inhibition activity was investigated with the synthesised compounds which showed promising inhibition. The K_Is were in the range of 54.6?nM–1.8?µM against hCA I, in the range of 32.1?nM–5.5?µM against hCA II and of 127?nM–2.12?µM against mtCA 3.     

Purification and some properties of L-glutamate decarboxylase from human brain

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000-fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N-terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 x 10(-3) M for glutamate and 0.13 x 10(-6) M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3-mercaptopropionic acid (Ki = 2.7 x 10(-6) M) while the Ki values for allylglycine and chloride ion are 1.8 x 10(-2) M and 2.2 x 10(-2) M, respectively.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Role of putrescine in enhancing shoot elongation in Scirpus mucronatus under submergence

Changes in polyamine biosynthesis in relation to submergence-enhanced shoot elongation were determined in shoots of Scirpus mucronatus L. Under submergence, the levels of free putrescine and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) increased, but the levels of free spermidine and spermine and the activity of S -adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) decreased. The increases in free putrescine and shoot elongation in submerged shoots diminished from the base to the apex. The increase in free putrescine in submerged shoots was coincident with the increase in shoot length. The submergence-induced increases in free putrescine and shoot elongation were inhibited by both 5 μ M a -difluoromethylarginine and 5 μ M a -difluoromethylornithine, and the inhibitory effects were reversed by 50 μ M putrescine. These overall results indicate that ADC- and ODC-mediated putrescine synthesis is essential for the elongation of Scirpus shoots grown under submergence.     

The use of steady-state rate equations to analyse progress curve data.

Analysis of progress curves for enzyme-catalyzed reactions has been made by using a procedure that does not require the derivation of complex integrated rate equations. The method involves conversion of progress curve data to reaction velocities that are then fitted to the appropriate differential rate equation. Application of the procedure to data obtained for the reaction catalyzed by aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), showed that the resulting values for the kinetic parameters agreed well with those obtained by conventional progress curve analysis (Duggleby, R.G. and Morrison, J.F. (1978) Biochim. Biophys. Acta 526, 398--409).     

Changes in L-ornithine decarboxylase activity during the cell cycle

The activity of L-ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17), the enzyme that catalyzes the initial and rate-limiting step in polyamine biosynthesis, has been studied in Chinese hamster ovary fibroblasts synchronized by selective detachment of mitotic cells. At various times after plating the distribution of cells among the G1, S and G2+M phases of the cell cycle was calculated from DNA distributions obtained by high-speed flow cytometric analysis. At these same times determination of the cellular L-ornithine decarboxylase activity showed that polyamine (putrescine) synthesis was initiated in mid-G1, that the rate of synthesis was maximal prior to DNA synthesis, and that it decreased during the S phase. A second increase in enzyme activity occurred before mitosis.     

S-adenosylmethionine decarboxylase from baker''s yeast.

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.     

Evaluation of Modified R-B System for Identification of Members of the Family Enterobacteriaceae

In a paired, double-blind study, the modified ("Beckford tube") R-B system was compared with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae from clinical isolates and stock cultures. The tests in the R-B system yielding positive reactions comparable to those predicted by Ewing's taxonomic classification of Enterobacteriaceae were production of hydrogen sulfide and presence of lysine and ornithine decarboxylasè activities. The test reactions in the R-B system found to be comparable to those in the conventional method were fermentation of glucose, hydrogen sulfide production, and lysine and ornithine decarboxylase activities. The production of gas from glucose was positive in the R-B system more often than in the conventional method; however, the motility test and the production of indole were positive less often in the R-B system. Adequate preliminary identification of the Enterobacteriaceae with the R-B system is enhanced if Simmons' citrate and Christensen's urea tests are used concomitantly. These findings emphasize the manufacturer's instructions that, in interpretation of results, colonial morphology and biochemical reactions must be used concurrently to make an accurate identification.     

Polyamines in various rice (Oryza sativa) genotypes with respect to sodium chloride salinity

Physiological responses of various rice genotypes were studied in relation to salt (NaCl) stress. Cultivars CSR-1 and Dular germinated well in different NaCl regimes compared to cvs Rupsail, Assam Getu and M-1–48. At 100 m M NaCl, the lowest germination was observed in cv. M-1–48. Cvs CSR-1 and Dular were relatively effective in maintaining high concentrations of polyamines as well as arginine decarboxylase (EC 4.1.1.19) activity in coleoptiles and roots in a non-stressed condition. The activities of two biodegradative enzymes, diamine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.4.3.4), were lowest in cv. CSR-1. The polyamine content was not significantly altered when seedlings of cv. CSR-1 were exposed to 100 m M NaCl. However, in cv. M-1–48 enhancement of arginine decarboxylase activity with concomitant accumulation of polyamines was observed. Leakage of metabolites and changes in the levels of Na⁺ and Cl⁻ were prominent in cv. M-1–48 under saline conditions. The results suggest a correlation between polyamine and salt stress-induced responses in rice genotypes.     

Decarboxylases for polyamine biosynthesis in Drosophila melanogaster larvae.

Ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17) and S-adenosyl-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lase, EC 4.1.1.50) were assayed in Drosophilia melanogaster larvae. The highest enzyme activities were detected in 24 and 48 h larvae, with diminishing activities in subsequent larval stages. Stimulation of S-adenosylmethionine decarboxylase by putrescine was demonstrable in late but not in early stages of larval development.     

Evaluation of the PathoTec ?Rapid I-D System” and Two Additional Experimental Reagent-Impregnated Paper Strips

The PathoTec "Rapid I-D System" and two experimental test strips for ornithine decarboxylase and beta-galactosidase have been evaluated for accuracy and ability to identify 1,252 members of the Enterobacteriaceae obtained from fresh clinical specimens. Accuracy of identification with the commercially available test system was 94.7%; this level increased to 98.5% with the addition of the two experimental test strips. Average individual accuracy of the 12-strip test system on a side-by-side basis with similar conventional procedures was 98%. In addition, 103 gram-negative nonfermentors were accurately grouped. The PathoTec System was applicable to 95% of the primary isolation plates used and provided biochemical data within 4 h after inoculation. The conventional test procedures were applicable to 100% of the primary isolation plates used and produced data within 48 h.     

AXONAL TRANSPORT OF CATECHOLAMINE SYNTHESIZING AND METABOLIZING ENZYMES

The rates of accumulation of the catecholamine synthesizing and metabolizing enzymes proximal to a ligation on the sciatic nerve of the rat were studied. Dopamine-β hydroxylase (EC 1.14.2.1) and tyrosine hydroxylase (EC 1.14.3a) accumulated at a similar rapid rate, and catechol-O-methyl-transferase (EC 2.1.1.6), choline acetyltransferase (EC 2.3.1.6) and monoamine oxidase (EC 1.4.3.4) accumulated at the same slow rate, whereas DOPA decarboxylase (EC 4.1.1.26) accumulated at an intermediate rate. Based on clearance of the rapidly accumulating enzymes, absolute flow rates were estimated to be: 106-167 mm/24 h for tyrosine hydroxylase; 138-185 mm/24 h for dopamine-β-hydroxylase; and 36-86 mm/24 h for DOPA decarboxylase. In contrast, the mean rate of transport of the slowly accumulating enzymes (monomine oxidase, catechol-O-methyltransferase and choline acetyltransferase) was approximately 3 mm/24 h. Colchicine and vinblastine completely blocked the axonal transport of both the rapidly and slowly transported enzymes. Studies of the subcellular distribution of each enzyme failed to confirm the suggestion that particulate enzymes are transported rapidly and soluble enzymes slowly. Our results suggest that the transport and inactivation of dopamine-β-hydroxylase, DOPA decarboxylase, and tyrosine hydroxylase are under different controls than monoamine oxidase and catechol-O-methyltransferase.