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Absorption of Ester Prodrugs in Caco-2 and Rat Intestine Models. Xin He, 2004.The aim of this study was to elucidate the absorption mechanism in Caco-2 and rat intestine models in order to improve the accuracy of prediction of oral absorption of ester prodrugs . Pivampicillin and cefcapene pivoxil hydrochloride (CFPN-PI), ester-type oral antibiotics, were chosen as model ester prodrugs . The level of esterase activity in Caco-2 cells was lower than that measured in the rat jejunum when p-nitrophenyl acetate was used as a substrate . Almost complete ester hydrolysis occurred before the ester prodrugs reached the basolateral side of the monolayer, and the disappearance of prodrugs was thought to be due to metabolism or transport after addition to the apical side of the monolayer . When pivampicillin and CFPN-PI were used, the amounts of ampicillin and cefcapene (CFPN) produced by hydrolysis of prodrugs were increased because intracellular degradation of prodrugs resulted in intracellular accumulation . On the other hand, when ampicillin or CFPN was used, only a small amount of the drug reached the basolateral side of the monolayers and no intracellular accumulation was observed . The permeability of CFPN-PI, the solubility of which is dependent on the acidity of gastric juice, across a Caco-2 monolayer or rat intestine, was also investigated by using an in vitro system that mimics the physiological state of the human gastrointestinal tract . The oral absorption of CFPN-PI in humans is predicted to be good either in the Caco-2 model or in the rat intestine model . It is concluded that our system may be a valuable tool for evaluation of oral absorption of ester prodrugs metabolized during permeation through the intestinal epithelium . Broader evaluation of such a system is warranted . Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii. Costanzo Bertoldo, 2004.The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii . In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced . The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima . The pullulanase gene from A . gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide . Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa . Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h . By using an alternative expression strategy in E . coli Tuner(DE3)(pLysS), the pullulanase gene from A . gottschalkii, including its signal peptide-encoding sequence, was cloned . In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg') . The N-terminal sequence of purified rPulAg' was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E . coli . Interestingly, most of the physicochemical properties of rPulAg' were identical to those of rPulAg . Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, ß-limited dextrins, and glycogen but not with amylose . This substrate specificity is typical of type I pullulanases . rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect . In addition, rPulAg' was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100 . The pullulanase from A . gottschalkii is the first thermoalkalistable type I pullulanase that has been described . Divergent Structure and Regulatory Mechanism of Proline Catabolic Systems: Characterization of the putAP Proline Catabolic Operon of Pseudomonas aeruginosa PAO1 and Its Regulation by PruR, an AraC/XylS Family Protein. Yuji Nakada, 2002.Pseudomonas aeruginosa PAO1 utilizes proline as the sole source of carbon and nitrogen via a bifunctional enzyme (the putA gene product) that has both proline dehydrogenase (EC 1.5.99.8) and pyrroline 5-carboxylate dehydrogenase (EC 1.5.1.12) activities . We characterized the pruR-putAP loci encoding the proline catabolic system of this strain . In contrast to the putA and putP (encoding proline permease) genes of other gram- negative bacteria, which are located at divergent or separate loci, Northern blotting demonstrated that the two genes form an operon in strain PAO1 . While the phylogenetic lineage of the PutP protein of strain PAO1 was related to that of the origin (80% identity to the P . putida counterpart), PutA of PAO1 (PutAPAO) was rather distantly related (47% identity) to the P . putida counterpart . Moreover, unlike the PutA proteins of P . putida and enteric bacteria, PutAPAO appeared to lack a regulatory function . Upstream of the putAP operon, the divergent PA0781 gene specified a hypothetical outer membrane protein with a molecular weight of 74,202 . This gene appeared to be dispensable for proline utilization as indicated by the normal growth of a knockout mutant of PA0781 on medium containing proline . The pruR (proline utilization regulator) gene immediately upstream of PA0781 encoded a transcriptional activator of the AraC/XylS protein family and mediated the proline-responsive expression of putAP. Primer extension studies identified a PruR-dependent promoter responsive to proline in the 5'-flanking region of putA. Thus, the proline utilization system of P . aeruginosa differs from that of P . putida with respect to putA structure, the organization of the putAP genes, and the regulatory mechanism of putA expression . Effect of aniA (Carbon Flux Regulator) and phaC (Poly-ß-Hydroxybutyrate Synthase) Mutations on Pyruvate Metabolism in Rhizobium etli. Michael F. Dunn, 2002.The Rhizobium etli poly-ß-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source . The inactivation of aniA, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate . Pyruvate carboxylase (PYC) activity, pyc gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58 . The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate . The possible relation between AniA, pyc transcription, and reduced-nucleotide levels is discussed . Positive Correlation between Tyrosine Phosphorylation of CpsD and Capsular Polysaccharide Production in Streptococcus pneumoniae. Matthew H. Bender, 2003.CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria . Using an immunoblotting technique, we observed distinct laddering patterns of S . pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size . Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall . Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers . The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased . Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern . However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments . In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D  P) correlated directly . In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rx1, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2DP levels . Thus, a factor outside the capsule locus, which is either missing or defective in the Rx1 background, is important in the control of capsule synthesis .
Microorganisms and Cancer: Quest for a Therapy. A. M. Chakrabarty, 2003.
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