Design,Synthesis, and In Vitro Evaluation of APIO Analogue of Neplanocin A

Abstract

A novel apio analogue of neplanocin A was efficiently synthesized from D-ribose via stereoselective aldol-retroaldol reaction for introducing hydroxymethyl group and RCM reaction for synthesizing carbocycle, and its inhibitory activity against SAH hydrolase was assayed.     

Role of active-site residues Thr81, Ser82, Thr85, Gln157, and Tyr158 in yeast cystathionine beta-synthase catalysis and reaction specificity

Cystathionine beta-synthase (CBS) effects the condensation of l-serine with l-homocysteine to form l-cystathionine. A series of active-site mutants, T81A, S82A, T85A, Q157A/E/H, and Y158F, was constructed to investigate effects on catalysis and reaction specificity in yeast CBS (yCBS). The effects of these mutations on the k(cat)/K(m)(L-Ser) for the beta-replacement reaction range from a reduction of only 3-fold for Y158F to below detectable levels for the Q157A and Q157E mutants. The order of importance of these residues to the beta-replacement reaction is Gln157 >or= Thr81 > Ser82 > Thr85 approximately Tyr158. All seven of the mutant enzymes catalyze a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH(3) and pyruvate. The ping-pong mechanism of CBS was thus expanded to include the latter reaction for these mutants. This activity is not detectable for wild-type yCBS, suggesting that the mutations result in a shift in the equilibrium between the open and the closed conformations of the active site of yCBS-substrate complexes. The Q157H and Y158F mutants additionally suffer suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme.     

Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor

A new metabolic pathway characterized recently that is expressed in activated macrophages involves the formation of nitric oxide ('N = O) as an intermediate. The 'N = O formed decomposes to nitrite (NO2-) and nitrate (NO3-). The substrate for the reaction is the amino acid arginine which is oxidized at the guanido nitrogen to yield citrulline as the other product of the reaction. The studies reported here show that the activity for this unusual oxidation reaction which is contained in the 100,000 x g supernatant was lost after desalting on a Sephadex G-25 column. A small molecule cofactor was found to be required for the restoration of activity. The addition of (6R)-tetrahydrobiopterin (BH4) and NADPH led to complete recovery of activity in this desalted protein. Analysis of macrophage cell extracts, using high performance liquid chromatography with electrochemical detection, showed that BH4 was present at 17 pmol/10(6) cells or 2.1 microM in macrophage supernatant. Only the (6R)-isomer was present. With the addition of BH4 and NADPH, there was loss of arginine that was equal to the NO2-, NO3-, and citrulline formed. With substoichiometric levels of NADPH relative to BH4, the loss of arginine was greater than the formation of the end products of the reaction. A scheme for the reaction pathway consistent with the results involves N-hydroxylation of arginine as the initial step. The participation of BH4 in this type of oxidative chemistry is consistent with previous characterizations of this co-factor.     

Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa.     

Active site labeling of the RNA polymerases A, B, and C from yeast

RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.     

The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane

The autofluorescent lipofuscin that accumulates in retinal pigment epithelial cells with age may contribute to an age-related decline in cell function. The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff base, N-retinylidene phosphatidylethanolamine from all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane; (ii) further reaction of N-retinylidene phosphatidylethanolamine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (iii) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogenic scheme, all-trans-retinal was reacted with dipalmitoyl-l-alpha-phosphatidylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass spectrometry revealing the molecular ion at m/z 1222.9 (C(77)H(124)O(8)PN) accompanied by product ion at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromatography peaks, each with UV similar to reference A2-PE. By mass spectrometry, A2-PE consisted of multiple peaks, representing fatty acids with different chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-PE as the A2E precursor.     

Shaken not stirred: a facile synthesis of 1,4-bis(furo[2,3-d]-pyrimidine-2,4(1H,3H)-dione-5-yl)benzenes by one-pot reaction of isocyanides, N,N'-dimethylbarbituric acid, and terephthaldialdehyde

A simple and efficient synthesis of 1,4-bis(furo[2,3-d]pyrimidine-2,4(1H,3H)-dione-5-yl)benzene derivatives was achieved via a one-pot three-component reaction of isocyanides, N,N'-dimethylbarbituric acid, and terephthaldialdehyde in DMF at room temperature for 30 min. These improved reaction conditions allow the preparation of highly substituted furopyrimidinones in high yields and purity under mild reaction conditions.     

Individual reaction requirements of two enzyme activities, isolated from Aspergillus parasiticus, which together catalyze conversion of sterigmatocystin to aflatoxin B1

Individual reaction requirements were determined for each of two enzyme activities present in Aspergillus parasiticus mycelia which together catalyze conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1). A postmicrosomal activity (PMA) catalyzed conversion of ST to O-methylsterigmatocystin (OMST) and a microsomal activity (MA) catalyzed conversion of OMST to AFB1. PMA was stimulated two- to three-fold in the presence of S-adenosylmethionine. Addition of NADPH promoted the maximum MA; this activity was not detected when FAD, FMN, NAD, or NADH were utilized individually as cofactors in reaction mixtures. A substantial amount (62%) of MA was lost during isolation of the microsomal fraction, but the activity was completely restored by reconstitution with a heat-treated (100 degrees C) postmicrosomal fraction. The reaction catalyzed by MA was optimum at pH 7.0 and at 17-23 degrees C, whereas the PMA reaction was optimum at pH 8.0-8.5 and at 35-40 degrees C. Apparent Km values of approximately 2.6 X 10(-6) M (for ST) and 6.6 X 10(-7) M (for OMST) were determined for PMA and MA, respectively.     

Biosynthesis of cholic acid in rat liver. 24-Hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid.

Conversion of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-[7beta-3H]cholestanoic acid into 3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta-cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 times g supernatant fluid or the microsomal fraction fortified with 100,000 times g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 times g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 times g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of 18O into the product when the reaction was performed in an atmosphere containing 18O2. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24-hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24alpha epimer of 3alpha, 7alpha,12alpha-trihydroxy-5beta-cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid was 24-hydroxylated more efficiently than the free acid.     

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro.

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.     

Stereoselectivity in reactions of amino acids catalyzed by pyridoxal derivatives carrying rigidly-attached chirally-mounted basic groups--transamination,racemization, decarboxylation,retro-aldol reaction,and aldol condensation

A tetrahydroquinoline ring was used to mount the critical functional groups of pyridoxal, and also two examples of rigidly held chirally mounted basic groups. They were able to selectively catalyze decarboxylation, aldol reaction, and retro-aldol reaction of amino acids rather than transamination, and with stereoselectivity. In the aldol reaction of glycine with acetaldehyde to synthesize threonine and allo-threonine, one of the catalysts reversed its stereoselectivity when the basic group was protonated. The observed stereoselectivities were all consistent with prediction.     

Localization of inhibin in testes of human, bonnet monkey, dog and rat by immunoperoxidase technique

Immunocytochemical study on the localization of inhibin in the testes of human, bonnet monkey, dog and rat was carried out using indirect immunoperoxidase technique, in order to investigate the cell types involved in inhibin production/storage. A positive reaction was observed in the testes of human, monkey and dog while it was negative in rat testis using specific antiserum to human testicular inhibin generated against homogeneous preparation of human testicular inhibin in our laboratory. Inhibin was found to be localized in Sertoli cells, spermatogonia and primary spermatocytes of human, monkey and dog testes. A weak positive reaction was observed in spermatids of human testis only. Interestingly, Leydig cells of human, monkey and dog testes showed positive reaction indicating presence of inhibin in these cells also.     

The effect of restricted hydration on the rate of reaction of glucose 6-phosphate dehydrogenase, phosphoglucose isomerase, hexokinase and fumarase. Relevance for metabolism in xeric (near-dry) conditions

A method is described for the measurement of enzyme activity under xeric conditions. The reaction mixtures had water contents ranging between 0.1 and 0.6g/g of reaction mixture. For glucose 6-phosphate dehydrogenase, hexokinase and fumarase, enzyme activity became detectable (about 0.05% of the fully hydrated rate) when the water content was about 0.2g/g of reaction mixture, and for phosphoglucose isomerase, around 0.15g/g of reaction mixture. With the water content raised to 0.3g/g of reaction mixture the reaction rates were only increased to 0.1-3% of the fully hydrated rate. When the combined rates for phosphoglucose isomerase and glucose 6-phosphate dehydrogenase were measured, reasonable agreement was found between the experimental data and those calculated from the individual experimentally determined rates on the assumption that diffusion was not further limiting. A method was devised for measuring the diffusion coefficients of low-molecular-weight substances in solutions having low water contents. The diffusion coefficients of riboflavin in sorbitol solution decreased by about 100-fold when the water content of the latter was reduced from 3 to 0.25g/g of sorbitol. It is concluded that to detect enzyme activity a certain minimal amount of water is required and that above this minimum the rate is still restricted by diffusion limitation. The relevance of the results to the physical state of water in reaction mixtures and to metabolism in seeds and spores in xeric conditions is discussed.     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.     

Preparation by grafting onto, characterization, and properties of thermally responsive polymer-decorated cellulose nanocrystals

The grafting of thermosensitive amine-terminated statistical polymers onto the surface of cellulose nanocrystals (CNCs) was achieved by a peptidic coupling reaction, leading to unusual properties like colloidal stability at high ionic strength, surface activity, and thermoreversible aggregation. We have used a large variety of experimental techniques to investigate the properties of the polymer-decorated CNCs at different length-scales and as a function of the different reaction parameters. A high grafting density could be obtained when the reaction was performed in DMF rather than water. Infrared and solid-state NMR spectroscopy data unambiguously demonstrated the covalent character of the bonding between the CNCs and the macromolecules, whereas TEM images showed a preserved individualized character of the modified objects. Dynamic light scattering and zeta potential measurements were also consistent with individual nanocrystals decorated by a shell of polymer chains. Surface tension measurements revealed that CNCs became surface-active after the grafting of thermosensitive amines. Decorated CNCs were also stable against high electrolyte concentrations. A thermoreversible aggregation was also observed, which paves the way for the design of stimuli-responsive biobased nanocomposite materials.     

Chlorogenate hydrolase-catalyzed synthesis of hydroxycinnamic acid ester derivatives by transesterification, substitution of bromine, and condensation reactions

A chlorogenate hydrolase (EC 3.1.1.42) synthesized 2-phenylethyl caffeate (2-CAPE) from 5-chlorogenic acid (5-CQA) and 2-phenylethyl alcohol (2-PA) (by transesterification), from 5-CQA and 2-phenylethyl bromide (2-PBr) (by substitution of bromine), and from caffeic acid (CA) and 2-PA or 2-PBr (by condensation) as well as hydrolysis of 5-CQA. Some reaction conditions including pH, temperature, substrate and solvent concentrates, and reaction time were optimized for the production of 2-CAPE. A maximal molar yield of 50% was achieved by transesterification, 4.7% by substitution of bromine, and 13% by condensation. Among the parameters studied for optimization, the pH of the buffer solution and concentration of 2-PA or 2-PBr affected the production of 2-CAPE. The optimum pH for the hydrolysis reaction was within the neutral range (pH 6.5), whereas the residual three reactions were only catalyzed within the acidic range (pH 3.0–4.0). The optimum concentrations of 2-PA and 2-PBr for three reactions were 5–70 vol% and no 2-CAPE was produced in the 2-PA or 2-PBr solutions containing powdered enzyme. The enzyme may bind to the caffeoyl moiety of 5-CQA or CA to form an enzyme–substrate complex. It then catalyzes four different reactions corresponding to the reaction conditions.     

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 x 10(2) and 2.5 x 10(5) cells g(-1) of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques.