Taste Masking Approaches for Medicines.

Undesirable taste is an important problem for patient compliance. Several oral pharmaceuticals and bulking agents have disagreeable and bitter-tasting components. The Bitter taste characteristics found in such systems have been eliminated or minimized by the development of numerous formulations with improved performance and acceptability especially in children and elderly patient. This review is mainly concerned with the bitter-masking techniques published in peer-reviewed journals and will shed light on different methods of masking undesirable taste of the drugs, and their pharmaceutical applications. Hence, nearly no reference will be made to the techniques published as patent applications.

Functional characterization of the polyketide synthase gene required for ochratoxin A biosynthesis in Penicillium verrucosum.

The ochratoxin A (OTA) polyketide synthase otapks gene has been cloned from Penicillium verrucosum. A P. verrucosum mutant in which the otapksPV gene has been interrupted cannot synthesize ochratoxin A. The protein is most similar to the citrinin polyketide synthase CtnpksMa from Monascus anka (83% identity at the amino acid level). Different nutritional conditions influence OTA production in P. verrucosum, with the addition of glycerol and galactose to MCB resulting in approximately 19 and 32 fold increases in OTA production respectively. These effects are mirrored in increased levels of otapksPV gene transcription. In contrast, the addition of glucose to MCB containing galactose results in an approximate 10 fold repression in OTA production, with this repression again being mirrored in decreased levels of otapksPV gene transcription. Thus the effects of different carbon sources on OTA production in P. verucosum appear to be regulated at the level of gene transcription. Two additional open reading frames, otaE and otaT, were identified in the 5' and 3' flanking regions of otapksPV, respectively. The otaT and otaE genes are co-expressed with P. verrucosum otapksPv, indicating a possible role for these genes in OTA biosynthesis. Furthermore, otaT and otaE were identified as putative homologues of the M. anka citrinin transporter ctnC (72% amino acid identity) and M. anka citrinin oxidoreductase ctnB (83% amino acid identity); suggesting that the genes involved in OTA production in P. verrucosum may be very similar to those involved in citrinin production in M. anka.

Analysis of the effect of nutritional factors on OTA and OTB biosynthesis and polyketide synthase gene expression in Aspergillus ochraceus.

The effect of a wide variety of nutritional based biotic factors on the production of both OTA and OTB biosynthesis in A. ochraceus was assessed. Different carbon sources including glucose, sucrose, maltose, galactose, xylose and glycerol appear to repress OTA production when the fungus is grown in OTA permissive PDY medium. In contrast lactose appears to induce OTA production, with the addition of lactose and galactose to the OTA restrictive PDC medium resulting in marked increases in OTA levels. The addition of lactose to MCB and PDY media considerably increases OTB production. The addition of both sucrose and galactose to MCB has similar yet less marked effects. Different nitrogen sources also affect OTA production with ammonium chloride significantly reducing OTA production, while organic nitrogen sources such as urea and amino acids including phenylalanine, lysine, glutamine and proline induce OTA production. The induction of otapksAo gene expression under these conditions correlates well with the levels of OTA produced under the same experimental conditions, suggesting that the observed effects appear to be modulated, at least in part, at the level of gene transcription. However while the levels of OTB produced in A. ochraceus also appear to be influenced by these nutritional based biotic factors, this appears to be regulated in a manner which is independent of otapksAo gene expression.

Characterisation of a pks gene which is expressed during ochratoxin A production by Aspergillus carbonarius.

Aspergillus carbonarius is considered the main fungus responsible for ochratoxin A (OTA) contamination in grapes. OTA is a potent nephrotoxin and a possible human carcinogen with a polyketide derived structure. Fungal polyketide synthases (PKSs) have recently been demonstrated to be involved in OTA biosynthesis in both Penicillium and Aspergillus species. We report here on the identification and characterisation of part of a novel polyketide synthase gene, ACpks from A. carbonarius. The sequence appears to encode conserved ketosynthase and acyl transferase domains, which are characteristic of previously characterised PKS enzymes. Expression of the ACpks gene is differentially regulated, with transcription being observed when the fungus was grown on synthetic grape medium and on OTA permissive medium (MM) whereas no transcription was detected when the fungus was grown on OTA restrictive medium (YES). ACpks expression was also observed when A. carbonarius was grown at low pH, with concomitant increases in OTA production. This correlation between ACpks gene expression and OTA production suggests the likely involvement for the product of this gene in ochratoxin A biosynthesis in the fungus. From a preliminary screening of Aspergillus isolates with ACpks specific primers, ACpks gene homologues appear to be present in A. sclerotioniger and A. ibericus, two species of section Nigri which are closely related to A. carbonarius.

Genetic analysis of genes involved in dipeptide metabolism and cytotoxicity in Pseudomonas aeruginosa PAO1.

The dipeptide transport operon in bacteria comprises genes for the transport and metabolism of amino acids and dipeptides, as well as haem and haem precursors such as aminolaevulinic acid. Such nutrient and mineral sources are vital for bacteria to survive in and colonize a range of niches. In silico analysis of the dipeptide transport systems in sequenced Pseudomonas species identified the presence of two genes in P. aeruginosa strains that were absent in other sequenced pseudomonads. These genes encode a putative metallopeptidase, PA4498, and a putative transcriptional regulator, PA4499. Proteomic profiling of wild-type PAO1 and a PA4499 mutant strain indicated that PA4499 negatively regulated the putative peptidase, PA4498. Transcriptional fusion analysis verified that expression of PA4498 (mdpA, metallo-dipeptidase aeruginosa) was negatively regulated by the downstream putative transcriptional regulator PA4499 (psdR, Pseudomonas dipeptide regulator). Transcriptional fusion analysis also showed that the dppABCDF operon was under the negative control of psdR. Functional genomic analysis of mdpA indicated that it is required for the metabolism of a range of dipeptides and that it contributes to the cytotoxicity of PAO1 on an epithelial cell line.

A role for TonB1 in biofilm formation and quorum sensing in Pseudomonas aeruginosa.

This study revealed that a Pseudomonas aeruginosa tonB1 mutant was unable to produce a mature biofilm and showed reduced swarming and twitching motilities compared with the parent strain. The tonB1 mutant was also found to produce significantly lower cell-free and cell-associated levels of the quorum sensing (QS) signal molecule 3-oxo-C12-AHL. Altered biofilm and motility phenotypes were restored to wildtype with the addition of exogenous N-acylhomoserine lactones. These functions were independent of the role of TonB1 in iron uptake. This is the first time that a link has been established between TonB1 activity and QS.

Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions.

Molecules exuded by plant roots are thought to act as signals to influence the ability of microbial strains to colonize the roots and to survive in the rhizosphere. Differential bacterial responses to signals from different plant species may mediate the selection of specific rhizosphere populations. Very little, however, is known about the effects of plant exudates on patterns of bacterial gene expression. Here, we have tested the concept that plant root exudates modulate expression of bacterial genes involved in establishing microbe-plant interactions. We have examined the influence on the Pseudomonas aeruginosa PA01 transcriptome of exudates from two varieties of sugarbeet that select for genetically distinct pseudomonad populations in the rhizosphere. The response to the two exudates showed only a partial overlap; the majority of those genes with altered expression was regulated in response to only one of the two exudates. Genes with altered expression included those with functions previously implicated in microbe-plant interactions, such as aspects of metabolism, chemotaxis and type III secretion, and a subset with putative or unknown function. Use of a panel of mutants with targeted disruptions allowed us to identify previously uncharacterized genes with roles in the competitive ability of P. aeruginosa in the rhizosphere within this subset. No genes with host-specific effects were identified. Homologues of the genes identified occur in the genomes of both beneficial and pathogenic root-associated bacteria, suggesting that this strategy may help to elucidate molecular interactions that are important for biocontrol, plant growth promotion, and plant pathogenesis.

Characterisation of the regulatory RNA RsmB from Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the molecular regulation of virulence factors and secondary metabolites is tightly controlled. This control involves several signal-mediated regulatory networks, including the GacS-GacA system and quorum sensing. Recently, the posttranscriptional repressor protein RsmA has been implicated in secondary metabolite production. RsmA is postulated to work in tandem with an as yet unidentified regulatory RNA molecule in a manner analogous to its homologues in other bacteria. Here we have identified a gene encoding an untranslated regulatory RNA (RsmB), located in the rpoS/ fdxA intergenic region of the P. aeruginosa PAO1 genome. Overexpression of rsmB in P. aeruginosa resulted in an increase in N-acyl-homoserine lactone, pyocyanin and elastase production compared with a marked decrease when rsmA was overexpressed. Mutation of rsmB resulted in a decrease in AHL production compared to wild type. We propose that RsmB is the cognate regulatory RNA of RsmA in P. aeruginosa. The global regulator GacA was not absolutely required for rsmB transcription in P. aeruginosa, as is the case in Pseudomonas fluorescens. However, GacA influenced the kinetics of rsmB transcription in that in late stationary phase the gacA mutant showed a substantial reduction in rsmB transcript levels compared to wild type. RsmA also influenced rsmB; in an rsmA mutant, the steady state level of rsmB transcript was reduced and this was due to a decrease in the transcription of rsmB. A balance in the levels of RsmA and RsmB may be an autoregulatory mechanism to ensure that RsmA is tightly controlled, as might be expected for such a potent global repressor.

The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance.

2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells.

Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113.

The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile. Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P. fluorescens F113 phlF mutant. The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a sigma(70) RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene. The repression by PhlF is due to its interaction with an inverted repeated sequence, phO, located downstream of the phlA transcriptional start site. Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind phO as a dimer or higher-order complex. Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to phO. PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-phO complex. Conversely, the PhlF-phO complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of phlA expression.