Pseudomonas aeruginosa alkaline protease can facilitate siderophore-mediated iron-uptake via the proteolytic cleavage of transferrins

In order to determine whether Pseudomonas aeruginosa alkaline protease AprA is involved in facilitating siderophore-mediated iron-acquisition from human transferrins, we measured bacterial growth, the production of siderophore and AprA, iron-acquisition from transferrins, and the proteolytic cleavage of transferrins in an alkaline minimal medium (pH 8.3) containing human transferrins as an iron source and compared these on a time scale. The growth of P. aeruginosa was found to be stimulated in proportion to the iron-saturation levels of transferrins. AprA production and the proteolytic cleavage of transferrins began concomitantly with siderophore production from the early growth phase when P. aeruginosa was actively growing and consuming most iron for growth. However, the AprA-free, but siderophore-containing, culture ultrafiltrates could also remove iron from transferrin. These results indicate that alkaline protease AprA can facilitate the siderophore-mediated iron-uptake of P. aeruginosa via the proteolytic cleavage of transferrins. However, the proteolytic cleavage by AprA is not essentially required for iron-acquisition from transferrins.     

Necessity of enzymatic activity of alkaline phosphatase for mineralization of osteoblastic cells

Alkaline phosphatase (ALP) is supposed to be important for bone formation; however, its role is not clear. In this study, we examined the importance of enzymatic activity of ALP and anchoring of ALP protein to the cells for mineralization of an osteoblastic cell line, MC3T3-E1. While we cultured the cells in the presence of tetramisole, an inhibitor of ALP activity, ALP protein was expressed at a similar level to that in the control. Although tetramisole showed no effect on cell growth and increased hydroxyproline accumulation, it decreased the osteocalcin production and the accumulation of calcium and phosphate in the matrices. Tetramisole also inhibited mineralized nodule formation, which was observed by optical microscopy and detected by Von Kossa staining. On the other hand, when ALP protein was released from the cell membranes with the use of phosphatidylinositol-specific phospholipase C, no marked changes were detected in hydroxyproline, calcium and phosphate accumulations in the matrices at late calcification stage, which was consistent with the morphological findings. These results clearly show that enzymatic activity of ALP is necessary for mineralization of MC3T3-E1 cells, but not the presence of ALP protein or anchoring of ALP to the cells.     

Cefazaflur: kinetics of hydrolysis in aqueous solution,acid dissociation constant and alkaline decomposition to fluorescent products

The values of the pseudo-first order hydrolysis rate constants in the pH range 1 to 10, and the pK_a, were determined for cefazaflur in aqueous solution at 37°C and ionic strength 0·2 M. A fluorimetric assay, based on alkaline hydrolysis at 100°C, was also developed for this compound. The results are consistent with previously reported related properties of other monoprotic cephalosporins.     

Chemical stability, enzymatic hydrolysis, and nasal uptake of amino acid ester prodrugs of acyclovir

The objective of this work was to improve nasal absorption of relatively impermeable small drug molecules via an amino acid prodrug approach. Acyclovir was selected as a model drug. L-Aspartate beta-ester, L-lysyl, and L-phenylalanyl esters of acyclovir were synthesized to investigate their effectiveness in enhancing nasal absorption of acyclovir. A stability study was conducted in phosphate buffer under various pH conditions at 25 and 37 degrees C. Enzymatic hydrolysis in rat nasal washings and plasma was conducted at 37 degrees C. A rat in situ nasal perfusion technique was utilized in this investigation to examine the rate and extent of nasal absorption of amino acid prodrugs. The remaining analyte concentrations in the nasal perfusate were quantitated by reversed-phase high-performance liquid chromatography. The results revealed that the L-lysyl and L-phenylalanyl esters were less stable than L-aspartate beta-ester. The stability of all three esters decreased with increasing pH and temperature. L-phenylalanyl ester is highly susceptible to plasma esterases, with an in vitro half-life 1.33 min. The rat in situ nasal perfusion study revealed that the extent of nasal absorption of acyclovir, L-lysyl and L-phenylalanyl esters was not significant (p < 1%). L-Aspartate beta-ester was absorbed to the extent of approximately 8% over 90 min of perfusion at an initial drug concentration of 100 microM. Nasal absorption of L-aspartate beta-ester of acyclovir was inhibited by L-asparagine but not by a dipeptide glycylsarcosine (Gly-Sar). The enhancement of acyclovir nasal absorption from the L-aspartate beta-ester prodrug suggests that nasal uptake of this prodrug probably involves an active transport system.     

Epimerization of benzylpenicilloic acid in alkaline media

5R,6R-Benzylpenicilloic acid was found to epimerize slowly in alkaline media to 5S,6R-benzylpenicilloic acid until equilibrium was established. Epimerization proceeded via the imine tautomer of penamaldic acid rather than the enamine form and was found to favor the 5S,6R-epimer at equilibrium. The conversion process was monitored using both reverse-phase high-performance liquid chromatography and NMR spectroscopy.     

Synthesis,docking, and in vitro studies of some substituted bischalcones on acid and alkaline phosphatases

A series of bischalcones was synthesized and screened for their effect on acid and alkaline phosphatases. In addition, molecular modeling and docking of these compounds into alkaline phosphatase using iGemdock was performed in order to predict the affinity and orientation of the designed compounds at the active site and was compared with the established inhibitor levamisole. The iGemdock docking fitness resulted in decrease in total energy (ranging from ?75.50 to ?100.84) for all the synthesized compounds which were less than levamisole (?50.69) revealing higher binding with the enzyme. The compounds were synthesized by Clasien–Schimdt condensation and their effect was observed on the activity of acid and alkaline phosphatases. The results showed that synthesized bischalcones were inhibitory to alkaline phosphatases, whereas an activating effect was observed on the activity of acid phosphatase. The type of inhibition and K _i values of bischalcones on alkaline phosphatase were also determined.     

Contributions of dam and conceptus to differences in sensitivity to valproic acid among C57 black and SWV mice

To ascertain the relative contributions of genotypes of conceptus and dam to developmental toxicity occasioned by valproic acid (VPA), crosses were established between resistant C57BL/6JBk (C, C57) and susceptible SWV/Bk (S, SWV) strains of mice. These included matings of pure lines, reciprocal outcrosses, and reciprocal backcrosses with F1 hybrids. At 8 d:12 h +/- 5 h, for each mating, 0, 500, or 600 mg/kg aqueous VPA was injected ip. Fetuses were examined on gestation day (gd) 18 for exencephaly (the paradigmatic anomaly), other abnormalities, mortality, litter size, and fetal weight. At 600 mg/kg, sensitivity to exencephaly induction in all cases was that of the dam, regardless of sire. Thus exencephaly here seems to be largely a function of the uterine environment produced by the maternal genotype. This inference is confirmed in backcrosses where F1-dams x S-sires and F1-dams x C-sires produced-identical outcomes, and S-dams x F1-sires produced much higher frequencies of exencephaly than C-dams x F1-sires. For prenatal mortality, the genotypes of both dam and conceptus appear to be important determinants. Fetal contribution is inferred from the observations that S-dam x S-sire matings produced a much higher frequency of mortality than S-dams x C-sires, and C-dams x C-sires produced higher mortality than C-dams x S-sires. Therefore, heterozygosity of the conceptus was protective. Among backcrosses, fetal determination of sensitivity to mortality is also seen by the observation that F1-dams x C-sires produces the same fetal mortality as C-dams x F1-sires. The contribution of uterine environment is seen in the observation that matings of S-dams x C-sires resulted in higher fetal mortality than did those with C-dams x S-sires. Therefore, identical conceptuses in different dams showed different levels of fetal loss. Thus exencephaly response appears to be largely controlled by genes active in the dam, and mortality as a result of a multigenic outcome with contributing genes active in both conceptus and dam. The data also suggest that SWV pure-line dams make a contribution to prenatal mortality not seen in C57 or F1 dams. Mean litter size among VPA-exposed litters showed high variability in pure lines and outcrosses. In backcrosses, F1 dams produced larger litters than pure line dams, arguing for heterosis as a contributor to this parameter. Reduction in litter size occasioned by VPA exposure was great in pure line dams and nonexistent in F1 dams. The SWV dams crossed with F1 sires were the only group among the backcrosses to show reduction of litter size, providing further confirmation of the increased sensitivity of pure-line (i.e., homozygous) SWV dams to VPA exposure. Fetal weight seems to be a function of uterine environment because female SWV produced conceptuses with lower fetal weight in all crosses, and produced a greater reduction in fetal weight attributable to VPA exposure than C57 or F1 dams. Fetal weight did not correlate closely with litter size, suggesting that a lower fetal weight may be a strain characteristic, as are exencephaly induction and prenatal mortality in response to VPA. Differences in sensitivity to VPA insult are seen for all parameters investigated with SWV dams being the most sensitive, but mechanisms seem to differ for a number of the endpoints.     

Oxidation of ochratoxin A by an Fe-porphyrin system: model for enzymatic activation and DNA cleavage

Ochratoxin A (OTA, 1) is a fungal toxin that facilitates single-strand DNA cleavage, DNA adduction, and lipid peroxidation when metabolically activated. To model the enzymatic activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) oxidation system. In its presence, OTA has been found to facilitate single-strand cleavage of supercoiled plasmid DNA through production of reactive oxygen species (ROS) (i.e., the hydroxyl radical, HO(*)). The reaction of OTA with the FeTPPS oxidation system also generated three hydroxylated products (chlorine atom still attached), which was taken as evidence for production of the known hydroxylated metabolites (2-4) of OTA. This result suggested that the FeTPPS system served as a reasonable model for the enzymatic activation of OTA. When the reaction of OTA with FeTPPS was carried out in the presence of excess hydrogen peroxide (H(2)O(2)) and sodium ascorbate, a hydroquinone species (OTHQ, 5) was detected in which an OH group has replaced the chlorine atom of OTA. The production of OTHQ (5) was dependent on the presence of the reducing agent, sodium ascorbate, which suggested that the oxidation catalyst furnished the quinone derivative OTQ (6) that was subsequently reduced to OTHQ (5) by ascorbate. Utilizing a synthetic sample of OTHQ (5), the hydroquinone was found to undergo autoxidation with a t(1/2) of 11.1 h at pH 7.4, and to possess a pK(a) value of 8.03 for the phenolic oxygen ortho to the carbonyl groups. Our findings imply that the hydroquinone (OTHQ) and quinone (OTQ) metabolites of OTA have the ability to cause alkylation/redox damage and have allowed us to propose a viable pathway for oxidative damage by OTA.     

Contributions to the development of laboratory equipment

The author's most important original constructive achievements over a period of about 25 years are reviewed within the framework of the three modern trends in the field of laboratory equipment: miniaturization, automation, and computerization. Mention is made of more than 20 devices, pieces of apparatus and installations, designed in order to solve some problems raised by the physico-chemical investigation of biological systems, which can likewise be used for paraclinical diagnosis in biochemistry and virology laboratories.