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PubMed1988

Clinical pharmacology of antibiotics and other drugs in cystic fibrosis.

The disposition of many drugs in cystic fibrosis is abnormal. In general, changes in pharmacokinetics include: increased volume of distribution, decreased plasma concentration, and enhanced renal and sometimes non-renal elimination of drugs. Pathophysiology of the disease important for drug disposition includes: (a) hypersecretion of gastric acid and duodenal secretions which are of small volume, viscous and low in bicarbonate; (b) increased intestinal permeability to some sugars and probe substances; (c) hypergammaglobulinaemia and sometimes hypoalbuminaemia; (d) significant elevation of free fatty palmitoleic acid level and decreased low-density and high-density serum lipoproteins; (e) an average increase by 30 to 45% in plasma volume in patients with cystic fibrosis who have moderately severe pulmonary disease, right ventricle hypertrophy and dilatation, which occurs in 15 to 35% of patients with a Shwachman score of 81 to 100; (f) abnormal bile acid metabolism and enterohepatic recirculation; and (g) enlarged kidneys and glomerulomegaly with increased glomerular filtration rate, tubular clearance and urine flow rate in some patients with cystic fibrosis. Delayed absorption from the gastrointestinal tract has been reported in patients with cystic fibrosis for cloxacillin, epicillin, clindamycin, ciprofloxacin and probably for cephalexin, para-aminobenzoic acid and chloramphenicol. A possible increased absorption was reported for cimetidine. Of 7 drugs studied only theophylline had significantly decreased plasma protein binding. An increased volume of distribution and increased renal clearance reported for several drugs is caused mainly by increases in plasma volume and urine flow rate in many of these patients. Possible increased elimination of some drugs in bile (which probably results from bile acid malabsorption) and in bronchial secretions (which are abundant in some cystic fibrosis patients with acute pulmonary infection) may explain enhanced non-renal elimination of these drugs. The metabolism of cimetidine in cystic fibrosis was reported not to be changed significantly compared to control subjects.

Anti-Bacterial Agents↗

PubMed2001

Capillary electrophoresis of sialic acid-containing glycoprotein. Effect of the heterogeneity of carbohydrate chains on glycoform separation using an alpha1-acid glycoprotein as a model.

alpha1-Acid glycoprotein (AGP) showed multiple peaks on separation using capillary electrophoresis in a chemically modified capillary with dimethylpolysiloxane at slightly acidic conditions. We analyzed glycoforms of AGP species after separation by ion-exchange chromatography, Con A affinity chromatography, and Cu(II)-chelating affinity chromatography. The AGP species thus obtained were digested with N-glycosidase F, and the released carbohydrate chains were analyzed by high-performance liquid chromatography after labeling with 3-aminobenzoic acid. The results afforded basic information on the contribution of carbohydrate chains to the separation mechanism of glycoforms of AGP by capillary electrophoresis. In addition, we describe an easy method for AGP analysis in serum samples using the electrokinetic injection.

Carbohydrate Sequence↗

PubMed1977

Chromatographic assays for folic acid.

TLC and high-pressure liquid chromatographic (HPLC) assays for folic acid were developed. In the TLC procedure, the folic acid band was extracted from the silica gel after development and determined spectrophotometrically by an oxidation and Bratton-Marshall sequence. A column packed with octadecylsilane chemically bonded to microparticulate silica gel was used for the HPLC assay. Potentiometric determination of water in folic acid samples was necessary to obtain accurate purity values. In a comparison of four assay methods, a direct colorimetric method gave precise, but unspecific, results, while the USP XIX colorimetric method lacked both precision and specificity. The two chromatographic methods were both precise and specific and gave the same assay results. Both methods separated p-aminobenzoic acid, N-(p-amino-benzoyl)-L-glutamic acid, 2-amino-1,4-dihydro-4-oxo-6-pteridinecarboxylic acid, 2-amino-4(1H)-pteridinone, and several unidentified impurities from folic acid.

Chromatography, High Pressure Liquid↗

PubMed1997

gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.

Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.

Azaserine↗

PubMed2006

Prodrug forms of N-[(4-deoxy-4-amino-10-methyl)pteroyl]glutamate-gamma-[psiP(O)(OH)]-glutarate, a potent inhibitor of folylpoly-gamma-glutamate synthetase: synthesis and hydrolytic stability.

Ester prodrugs of the phosphinate pseudopeptide N-[(4-deoxy-4-amino-10-methyl)pteroyl]glutamate-gamma-[psiP(O)(OH)]-glutarate (1a) were synthesized. H-phosphinic acids derived from N-Cbz vinyl glycine esters were converted to the desired pseudopeptides by Michael addition to alpha-methyleneglutarate esters. Pivaloyloxymethyl (POM) ester moieties were incorporated in both the N-terminal and C-terminal fragments prior to formation of either C-P bond. N-Alkylation of the corresponding amides derived from p-(N-methyl)aminobenzoic acid with 2,4-diamino-6-(bromomethyl)pteridine gave the target compounds. POM esters of methotrexate and the corresponding gamma-glutamyl conjugate were also synthesized using the same strategy. All prodrugs were evaluated in Chinese hamster ovary cells. Although the pseudopeptide prodrugs were ineffective, prodrugs of methotrexate and the corresponding gamma-glutamyl conjugate were equipotent with the parent compounds. Stability of the prodrugs was investigated in both phosphate buffer and cell line medium to provide a rationale for the observed biological data.

Aminopterin↗

PubMed1972

Tentative method for the qualitative detection and quantitative assessment of air contamination by drugs.

A method for detecting and measuring air contamination by drugs is described which uses an electrostatic bacterial air sampler, sprayers for micronizing drugs, and Mueller-Hinton medium seeded with a highly susceptible strain of Sarcina lutea. Three antibiotics (penicillin, tetracycline, aminosidine) and a sulfonamide (sulfapyrazine) were identified by pretreating portions of medium, showing no bacterial growth, with penicillinase or p-aminobenzoic acid solution and subsequently determining how both drug(-) susceptible and drug-resistant strains of Staphylococcus aureus were affected by this pretreatment. Quantitative determinations were also attempted by measuring the size of the inhibition zones.

Air↗

PubMedDate not supplied

[Synthetic medium for culturing Clostridium sporogenes].

Clostridium sporogenes was shown to be capable of good stable-growth and biomass accumulation up to 5 mg/ml (dry weight) in a chemically defined medium. Along with glucose, the culture required the following amino acids for its growth: arginine, phenylalanine, glycine tyrosine, leucine, isoleucine, valine, tryptophan, and methionine. The culture could grow in the absence of inorganic nitrogen but required such elements as phosphorus, iron and magnesium. Addition of cysteine to the medium accelerated the growth and increased the biomass yield. Leucine at a concentration over 0.1% inhibited the growth. Two vitamins, viz. biotin and p-aminobenzoic acid, are sufficient for the growth of the culture.

Amino Acids↗

PubMed1997

Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides.

The substrate specificity of the major cysteinyl proteinase of the parasitic protozoan Trypanosoma cruzi (cruzipain) was investigated, by combinatorial replacement of amino acid residues at positions P5-P'5, using a fluorescent quenched solid-phase library assay. Positively charged residues appear to be a general preference in the P5-P3 and the P'5-P'3 positions, while a hydrophobic residue was always required at the P2 position. A broad range of amino acids could be accepted at the P'1 position. A clear difference in terms of specificity between cruzipain and human cathepsin L was observed for the accommodation of Pro at the P2 position. The P1 specificity was investigated by a more detailed enzyme kinetic analysis using peptidyl-MCA (where MCA is methylcoumarin amide) and Abz-peptidyl-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] as substrates, and the results were compared with those obtained using human cathepsin L. Cruzipain showed a clear preference for benzyl-Cys or Arg at the P1 position. Human cathepsin L presented similar behaviour to that of cruzipain for the hydrolysis of the epsilon-NH2-Cap-Leu-Xaa-MCA (where Cap is epsilon-aminocaproyl) and Abz-Lys-Leu-Xaa-Phe-Ser-Lys-Gln-EDDnp series, whereas the mammalian enzyme was able to tolerate large P1 residues, such as phenylalanine, better than cruzipain in the latter series.

Alanine↗

PubMed1982

Investigations on nonsulfonamide Bratton-Marshall-positive compounds in porcine liver.

An unknown Bratton-Marshall-positive compound was observed when liver extracts were screened by a procedure for the simultaneous determination of sulfamethazine and sulfathiazole in swine livers. The unknown has been conclusively identified as o-aminobenzoic acid (anthranilic acid), a tryptophan metabolite, by gas-liquid chromatography-mass spectrometry of swine liver extracts obtained by a modified Tishler procedure for sulfonamide residues in animal tissues.

Animals↗

PubMed1992

Analogs of Ac-CCK-7 incorporating dipeptide mimics in place of Met28-Gly29.

A series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met28-Gly29-Trp-Met-Asp- Phe-NH2, (1)] were prepared in which the Met28-Gly29 dipeptide was replaced by omega-aminoalkanoic acids. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors, respectively, and for anorectic activity after intraperitoneal administration to rats. The analog incorporating 4-aminobutanoic acid (5) was only 8 times less potent than 1 in the pancreatic binding assay, was more potent in the striatal binding assay, and was more potent than 1 in reducing food intake in rats. Using a bioactive cyclic analog of Ac-CCK-7 as a template, several rigid spacers were designed and tested as substitutes for the Met28-Gly29 dipeptide. The analogs incorporating 3-aminobenzoic acid (20) and (1S)-trans-2-aminocyclopentanecarboxylic acid (26) proved highly effective in the binding assays and as anorectic agents. We hypothesize that for stimulation of CCK-A receptors, the main function of the N-terminal tripeptide of Ac-CCK-7 is to orient the tyrosine sulfate with respect to Trp30 and that the bioactive arrangement of these elements lies among those which are readily available to both 20 and 26. NOESY and distance-constrained molecular dynamics experiments carried out on 20 and 26 identified conformations in which the relative orientation of the tyrosine hydroxide and the alpha-carbon atom of tryptophan were similar, providing the basis for further drug design efforts.

Amino Acid Sequence↗

PubMed1984

Photoaffinity labelling of dopamine receptors. Synthesis and binding characteristics of azapride.

A new compound, azapride, i.e. 4-azido-5-chloro-2-methoxy-N-[1-(phenylmethyl)-4-piperidinyl]benza mide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation-induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation-induced inactivation by azapride remained unchanged in the presence of the scavenger p-aminobenzoic acid. These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors.

Affinity Labels↗

PubMed1994

Photoreactivity of chloramphenicol in vitro and in vivo.

The negative side effects of chloramphenicol (CAP) mostly involve blood dyscrasias (e.g. irreversible non-dose-dependent aplastic anemia), allergic skin reactions and eye damage. To learn the cause of these side effects, most research focuses on metabolically formed nitroso- and hydroxylamino derivatives in the predisposed patient. In previous investigations it was demonstrated that photochemical decomposition of CAP in vitro by UV-A leads to formation of p-nitrobenzaldehyde (pNB), p-nitrobenzoic acid (pNBA) and p-nitrosobenzoic acid (pNOBA); the latter comprises up to 45 mol% of the starting amount of CAP. Incubation of these photoproducts in rat blood showed that pNB and pNOBA rapidly react and that PNBA is stable under these conditions. Reaction products from pNB (half-life 1.7 min) proved to be pNBA and p-nitrobenzyl alcohol (pNBOH) while pNOBA (half-life 3.7 min) was converted into p-aminobenzoic acid (pABA). Exposure of CAP in rat blood to UV-A yielded the same end products: pNBA, pABA and pNBOH. To estimate the amount of oxidative stress generated in vivo by these compounds, the ability to form methemoglobin (MetHb) in erythrocytes was tested; only pNOBA and p-hydroxylaminobenzoic acid (pHABA), a possible intermediate in the decomposition of pNOBA, proved to be reactive. Ultraviolet-A exposure of rats, after intraperitoneal injection of CAP, led to 3.6 times the basic level of MetHb. In addition, covalent binding of 3H-labeled CAP photoproducts to the skin of the back and to the ears was found, which was 9.1 and 3.2 times higher, respectively, than the dark values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid.

The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H(2)O(2)-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 10(6) cells/ml media) with H(2)O(2) (0-200 microM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 microM H(2)O(2). Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 microM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 microM H(2)O(2). Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H(2)O(2). Addition of high concentrations of H(2)O(2) (200 microM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H(2)O(2) itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.

Antioxidants↗

PubMed2003

Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein.

The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o -aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S(1)' subsite, with a strong preference for aspartate. Subsites S(2)', S(1) and S(2) exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P(2)', or valine, isoleucine or histidine in P(1) precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P(2) to P(2)' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/ K(m) of 167 mM(-1) x s(-1). In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N -(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity.

Amino Acid Sequence↗

PubMed1992

Side chain modified 5-deazafolate and 5-deazatetrahydrofolate analogues as mammalian folylpolyglutamate synthetase and glycinamide ribonucleotide formyltransferase inhibitors: synthesis and in vitro biological evaluation.

5-Deazafolate and 5-deazatetrahydrofolate (DATHF) analogues with the glutamic acid side chain replaced by homocysteic acid (HCysA), 2-amino-4-phosphonobutanoic acid (APBA), and ornithine (Orn) were synthesized as part of a larger program directed toward inhibitors of folylpolyglutamate synthetase (FPGS) as probes of the FPGS active site and as potential therapeutic agents. The tetrahydro compounds were also of interest as non-polyglutamatable inhibitors of the purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFT). Reductive coupling of N2-acetamido-6-formylpyrido[2,3-d]pyrimidin-4(3H)-one with 4-aminobenzoic acid, followed by N10-formylation, mixed anhydride condensation of the resultant N2-acetyl-N10-formyl-5- deazapteroic acid with L-homocysteic acid, and removal of the N2-acetyl and N10-formyl groups with NaOH, afforded N-(5-deazapteroyl)-L-homocysteic acid (5-dPteHCysA). Mixed anhydride condensation of N2-acetyl-N10-formyl- 5-deazapteroic acid with methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid, followed by consecutive treatment with Me3SiBr and NaOH, yielded D,L-2-[(5-deazapteroyl)amino]-4-phosphonobutanoic acid (5-dPteAPBA). Treatment with NaOH alone led to retention of one ethyl ester group on the phosphonate moiety. Catalytic hydrogenation of N2-acetyl-N10-formyl-5-deazapteroic acid followed by mixed anhydride condensation with methyl L-homocysteate and deprotection with NaOH afforded N-(5,6,7,8-tetrahydro-5-deazapteroyl)-L-homocysteic acid (5-dH4PteHCysA). Similar chemistry starting from methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid and methyl N delta-(benzyloxycarbonyl)-L-ornithinate yielded D,L-2-[(5-deaza-5,6,7,8-tetrahydropteroyl)amino]-4-phosphonobut ano ic acid (5-dH4Pte-APBA) and N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine (5-dH4PteOrn), respectively. The 5-deazafolate analogues were inhibitors of mouse liver FPGS, and the DATHF analogues inhibited both mouse FPGS and mouse leukemic cell GARFT. Analogues with HCysA and monoethyl APBA side chains were less active as FPGS inhibitors than those containing an unesterified gamma-PO(OH)2 group, and their interaction with the enzyme was noncompetitive against variable folyl substrate. In contrast, Orn and APBA analogues obeyed competitive inhibition kinetics and were more potent, with Ki values as low as 30 nM. Comparison of the DATHF analogues as GARFT inhibitors indicated that the Orn side chain diminished activity relative to DATHF, but that the compounds with gamma-sulfonate or gamma-phosphonate substitution retained activity, with Ki values in the submicromolar range. The best GARFT inhibitor was the 5-dH4PteAPBA diastereomer mixture, with a Ki of 47 nM versus 65 nM for DATHF. None of the compounds showed activity against cultured WI-L2 or CEM human leukemic lymphoblasts at concentrations of up to 100 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗

PubMedDate not supplied

Volatile fatty acid-sensing system involving coenzyme-A transferase.

On-site monitoring of volatile fatty acids (VFAs), such as propionate, is industrially and medically important. The present study developed a VFA biosensing system comprised of two recombinant enzymes, propionate coenzyme A (CoA) transferase (PCT) from Clostridium propionicum and acyl-CoA oxidase from Arabidopsis thaliana. This system produced hydrogen peroxide in the presence of acetyl-CoA, oxygen, and VFA substrates, which could be quantified by colorimetric methods using peroxidase and dye reagents (e.g., p-aminobenzoic acid plus 4-aminoantipyrine or Amplex Red). The use of PCT and acetyl-CoA, rather than acyl-CoA synthetases (ACS) and CoA-SH, obviated a background reaction of dye reagents with CoA-SH and enabled very sensitive detection of VFAs (down to 1 microM propionate, more than 100-fold more sensitive compared to previously developed ACS biosensors). We demonstrated its utility by measuring propionate concentrations in serum and fermentation samples. Results suggest that our biosensing system is applicable to the detection of propionate in medical and fermentation samples.

Animals↗

PubMed1998

Intracellular multiplication and virulence of Shigella flexneri auxotrophic mutants.

We have constructed and analyzed a group of Shigella flexneri 5 auxotrophic mutants. The wild-type strain M90T was mutagenized in genes encoding enzymes involved in the synthesis of (i) aromatic amino acids, (ii) nucleotides, and (iii) diaminopimelic acid. In this way, strains with single (aroB, aroC, aroD, purE, thyA, and dapB) and double (purE aroB, purE aroC, purE aroD, purE thyA) mutations were obtained. Although the Aro mutants had the same nutritional requirements when grown in laboratory media, they showed different degrees of virulence in vitro and in vivo. The aroB mutant was not significantly attenuated, whereas both the aroC and aroD strains were severely attenuated. p-Aminobenzoic acid (PABA) appeared to be the main requirement for the Aro mutants' growth in tissue culture. Concerning nucleotides, thymine reduced the pathogenicity, whereas adenine did not. However, when combined with another virulence-affecting mutation, adenine auxotrophy appeared to potentiate that mutation's effects. Consequently, the association of either the purE and aroC or the purE and aroD mutations had a great effect on virulence as measured by the Sereny test, whereas the purE aroB double mutation appeared to have only a small effect. All mutants except the dapB strain seemed to move within a Caco-2 cell monolayer after 3 h of infection. Nevertheless, the auxotrophs showing a high intracellular generation time were negative in the plaque assay. Knowledge of each mutation's role in attenuating Shigella strains will provide useful tools in designing vaccine candidates.

Adenine↗

PubMedDate not supplied

Aromatic-dependent Salmonella as live vaccine presenters of foreign epitopes as inserts in flagellin.

Synthetic oligonucleotides specifying amino acid sequences identified as epitopes of various foreign antigens (cholera toxin subunit B, hepatitis B surface protein and others) have been inserted at an EcoRV-EcoRV deletion site in a cloned Salmonella flagellin gene; the resulting plasmids, when placed in flagellin-negative Escherichia coli or Salmonella sp. strains, caused production of flagellin expressing the epitope. If the chimeric flagellin allowed formation of flagella, the epitope was exposed at the surface of the flagellar filaments. A delta aroA flagellin-negative S. dublin live vaccine strain given plasmids carrying various chimeric flagellin genes was administered to mice, etc. Serum antibody specific for the foreign epitope was in all cases evoked by parenteral administration; oral route administration was effective in the case of two epitopes of hepatitis B surface protein but not effective for several other epitopes. Several i.p. inocula of the live vaccine strain with an insert corresponding to the 15 N-terminal amino acids of the M protein of Streptococcus pyogenes type 5 evoked M-specific antibody with opsonic activity, and the mice were (incompletely) protected against a lethal challenge of S. pyogenes type 5. The non-virulence of Salmonella sp. strains with complete blocks in the aromatic biosynthesis pathway, even for animals with genetically determined or other defects in host defences, can be completely accounted for by their requirement for p-aminobenzoic acid, since non-leaky pabB mutations caused similar attenuation. Two transposon insertions at aroE caused little or no attenuation, presumably because they did not result in complete block of the relevant step in biosynthesis. The limited growth of delta aroA strains in mouse tissues parallels that which precedes the bacteriostasis caused by addition of a sulphonamide to a growing broth culture of a sulphonamide-sensitive strain; the final cessation of growth in each case presumably results from inability to initiate new protein chains with a formyl-methionine unit when the original folic acid content of the bacteria has been diluted out by residual growth.

Animals↗