Characterization of folate production and probiotic potential of Streptococcus gallolyticus subsp. macedonicus CRL415.

Mandatory fortification of foods with folic acid is being questioned by many scientists principally because of the potential adverse secondary effects associated with their excessive consumption. It has been shown that selected strains of lactic acid bacteria (LAB) are able to produce natural forms of folate and these could be included in foods to prevent deficiencies without causing adverse effects. The aim of this study was to evaluate folate production and fol gene expression by Streptococcus gallolyticus subsp. macedonicus (S. macedonicus) CRL415 under different growth conditions in vitro and to assess its potential probiotic application. Results showed that glucose as the principal carbon source, and incubation at 42 °C under controlled pH conditions (6.0) increased folate production, fol gene expression, and growth of S. macedonicus CRL415. This strain was able to produce elevated folate concentrations during milk fermentation without the need of prolonged incubation times and was able to resist conditions simulating the gastrointestinal tract. In addition, S. macedonicus was susceptible to all required antibiotics, and had a good adhesion level to intestinal cells in vitro, making it a promising candidate for biotechnological application as functional starter cultures in the dairy industry.

Benzenesulfonamides act as open-channel blockers on KV3.1 potassium channel.

KV3.1 blockers can serve as modulators of the rate of action potential firing in neurons with high rates of firing such as those of the auditory system. We studied the effects of several bioisosteres of N-alkylbenzenesulfonamides, and molecules derived from sulfanilic acid on KV3.1 channels, heterologously expressed in L-929 cells, using the whole-cell patch-clamp technique. Only the N-alkyl-benzenesulfonamides acted as open-channel blockers on KV3.1, while molecules analogous to PABA (p-aminobenzoic acid) and derived from sulfanilic acids did not block the channel. The IC50 of six N-alkyl-benzenesulfonamides ranged from 9 to 55 µM; and the Hill coefficient suggests the binding of two molecules to block KV3.1. Also, the effects of all molecules on KV3.1 were fully reversible. We look for similar features amongst the molecules that effectively blocked the channel and used them to model a blocker prototype. We found that bulkier groups and amino-lactams decreased the effectiveness of the blockage, while the presence of NO2 increased the effectiveness of the blockage. Thus, we propose N-alkylbenzenesulfonamides as a new class of KV3.1 channel blockers.

Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p < 0.0001). This highly significant correlation was maintained for samples with the NAT1*4 (p = 0.002) and NAT1*14B haplotypes (p = 0.0106). These results provide the first documentation that NAT1-catalyzed N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

Genotoxicity (mitotic recombination) of the cancer chemotherapeutic agents cisplatin and cytosine arabinoside in Aspergillus nidulans.

Cisplatin (cis-diamminedichloroplatinum, cis-DDP) and cytosine arabinoside (ara-C) are anticancer drugs used in the treatment of human cancer. The two chemotherapeutic drugs were tested in current research for their recombinogenic potential in diploid cells of Aspergillus nidulans. Non-cytotoxic concentrations of ara-C (0.4 and 0.8 microM) and cis-DDP (1.5, 3.0 and 6.0 microM) were strong recombinagens in A. nidulans UT448//A757 diploid strain, which induced homozygosis of recessive genetic markers, previously present in heterozygous condition. Drugs significantly increased homozygosity index (HI) values for five nutritional genetic markers when compared with those determined in the absence of anticancer drugs. Since mitotic recombination is a mechanism leading to malignant growth through loss of heterozygosity at tumor-suppressor loci, ara-C and cis-DDP may be characterized as secondary promoters of malignant neoplasia in diagnosed cancer patients, after chemotherapy treatment.

p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase.

Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3.8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.

Fluorescence study of conformational properties of melanotropins labeled with aminobenzoic acid.

The native hormone alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analog [Nle(4),D-Phe(7)]alpha-MSH (NDP-alpha MSH), labeled at the amino terminal with the fluorescent aminobenzoic acid (Abz) isomers, were examined by fluorescence methods. We observed energy transfer between the tryptophan(9) residue acting as donor and Abz as acceptor, the transfer being more pronounced to the ortho-form of the acceptor. Within the hypothesis that different peptide conformations coexist in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function f(r). From the time-resolved fluorescence experimental data, we recovered the distance distribution between Abz and Trp(9), using the CONTIN program, within the framework of the Förster resonance energy transfer model. The methodology proved to be useful to provide quantitative information about conformational dynamics of melanotropins and its dependency on the solvent. In aqueous medium, alpha-MSH has a broad Abz-Trp(9) distance distribution, reflecting the structural flexibility of the peptide. Three different distance populations could be identified in the labeled analog NDP-alpha MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abz-Trp(9) distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides.

Bentiromide test using liquid-chromatographic measurement of p-aminobenzoic acid and its metabolites for diagnosing pancreatic insufficiency in childhood.

We assessed the diagnostic capability of the bentiromide test using a high-pressure liquid-chromatography method to analyze p-aminobenzoic acid and its metabolites in plasma as an indirect measure of exocrine pancreatic function. Mean total amine concentration in pancreatic-insufficient subjects was significantly lower than in control subjects. There were 3 of 15 false-negative results and no false-positive results. We conclude that this chromatographic method is an effective means of analyzing p-aminobenzoic acid and its metabolites after ingestion of bentiromide.

Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase.

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.