Modeling non-inherited antibiotic resistance.

A mathematical model is presented for the increase and decrease of non-inherited antibiotic resistance levels in bacteria. The model is applied to experimental data on E. coli exposed to amoxicillin or tetracyclin in different concentrations. The parameters of the model are estimated using a Monte Carlo Markov Chain method. The model accurately describes build-up and decline of antibiotic resistance caused by physiological adaptations as long as no genetic changes have occurred. The main conclusion of the analysis is that short time periods are sufficient to re-obtain low MIC-values after long-lasting exposure to these antibiotics.

Water extracts of cabbage and kale inhibit ex vivo H(2)O(2)-induced DNA damage but not rat hepatocarcinogenesis.

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito's hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 +/- 7.8 microm) and kale- (110.8 +/- 10.0 microm) treated animals compared with control (120.9 +/- 12.7 microm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 +/- 0.3 microg/g) or kale (4 +/- 0.2 microg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 +/- 0.07 microg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Evaluation of DNA damage by the alkaline comet assay of the olfactory and respiratory epithelia of dogs from the city of São Paulo, Brazil.

Animals kept as pets may be considered sentinels for environmental factors to which humans could be exposed. Olfactory and respiratory epithelia are directly subjected to airborne factors, which could cause DNA lesions, and the alkaline comet assay is considered a reliable tool for the assessment of DNA damage. The objective of this work is to evaluate the extent of DNA damage by the comet assay of the olfactory and respiratory epithelia of dogs from different regions of the city of São Paulo, Brazil. Thirty-three clinically healthy dogs, aged 5 years or more, were used in the study, with 7 from the North region of São Paulo, 7 from the South region, 3 dogs from the East region, and 16 dogs from the West city region. Three dogs younger than 6 months were used as controls. DNA damage was analyzed by the alkaline comet assay. We observed no difference in histopathological analysis of olfactory and respiratory epithelia between dogs from different regions of São Paulo. Dogs older than 5 years presented significantly higher comet length in both olfactory and respiratory epithelia, when compared with controls, indicating DNA damage. When separated by regions, olfactory and respiratory epithelia presented similar DNA damage in dogs from different regions of São Paulo, corroborating with similar levels of particulate matter index (PM10) in all regions of the city. In this study, we report for the first time that the comet assay can be used to quantify the extent of DNA damage in dog olfactory and respiratory epithelia, and that comet length (DNA damage) increases with age, probably due to environmental factors. Air pollution, as measured by PM10, can be responsible for this DNA damage.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein.

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

How light-induced charge transfer accelerates the receptor-state recovery of photoactive yellow protein from its signaling state.

Stark (electroabsorption) spectra of the M100A mutant of photoactive yellow protein reveal that the neutral, cis cofactor of the pB intermediate undergoes a strikingly large change in the static dipole moment (|Deltamu| = 19 Debye) on photon absorption. The formation of this charge-separated species, in the excited state, precedes the cis --> trans isomerization of the pB cofactor and the regeneration of pG. The large |Deltamu|, reminiscent of that produced on the excitation of pG, we propose, induces twisting of the cis cofactor as a result of translocation of negative charge, from the hydroxyl oxygen, O1, toward the C7-C8 double bond. The biological significance of this photoinduced charge transfer reaction underlies the significantly faster regeneration of pG from pB in vitro, on the absorption of blue light.

From primary photochemistry to biological function in the blue-light photoreceptors PYP and AppA.

To properly respond to changes in fluency conditions, Nature has developed a variety of photosensors that modulate gene expression, enzyme activity and/or motility. Dedicated types have evolved, which can be classified in six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins and BLUF-proteins. The photochemistry of the first three families is based on cis/trans isomerization of an ethylene bond. Surprisingly, the latter three all use flavin as their chromophore, but each with very different photochemistry. In this contribution we will discuss the molecular basis of signal generation in a xanthopsin (Photoactive Yellow Protein (PYP) from Halorhodospira halophila), a photoreceptor for negative phototaxis, and in a BLUF protein (AppA from Rhodobacter sphaeroides), a transcriptional anti-repressor. PYP is activated through trans/cis isomerization of the 7,8-vinyl bond of its 4-hydroxycinnamic acid chromophore. This initiates a photocycle with multiple intermediates, like pB, which is formed after intramolecular proton transfer. The negative charge thus formed in the interior of the protein triggers formation of a partially unfolded signaling state. For AppA much less is known about the underlying photochemistry. Available evidence suggests that it is based on a light-induced change in the hydrogen-bonding of its flavin chromophore and/or a change in hydrophobic stacking between the flavin and/or nearby aromatic amino acids like Y 21. A signaling state is formed within microseconds, which recovers with a rate of approximately 10(-3) s(-1). The change in conformation between receptor- and signaling-state in AppA, however, appear to be minute as compared to those in PYP. Here we review the underlying chemistry in the various steps of the photocycle of these two photoreceptor proteins and provide new data on their mechanism and function.

Stark spectroscopy on photoactive yellow protein, E46Q, and a nonisomerizing derivative, probes photo-induced charge motion.

The change in the electrostatic properties on excitation of the cofactor of wild-type photoactive yellow protein (WT-PYP) have been directly determined using Stark-effect spectroscopy. We find that, instantaneously on photon absorption, there is a large change in the permanent dipole moment, /Delta(-->)mu/, (26 Debye) and in the polarizability, (-)Deltaalpha, (1000 A(3)). We expect such a large degree of charge motion to have a significant impact on the photocycle that is associated with the important blue-light negative phototactic response of Halorhodospira halophila. Furthermore, changing E46 to Q in WT-PYP does not significantly alter its electrostatic properties, whereas, altering the chromophore to prevent it from undergoing trans-cis isomerization results in a significant diminution of /Delta(-->)mu/ and (-)Deltaalpha. We propose that the enormous charge motion that occurs on excitation of 4-hydroxycinnamyl thioester, the chromophore in WT-PYP, plays a crucial role in initiating the photocycle by translocation of the negative charge, localized on the phenolate oxygen in the ground state, across the chromophore. We hypothesize that this charge motion would consequently increase the flexibility of the thioester tail thereby decreasing the activation barrier for the rotation of this moiety in the excited state.

The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation.

It is shown that the N-terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N-terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N-terminal domain is linked through a hydrogen-bonding network with the chromophore-containing domain of the protein. Furthermore, deletion of the N-terminal domain completely abolishes the non-linearity of the Arrhenius plot of the rate of ground state recovery.

Association of attending physician specialty with the cesarean delivery rate in the same patient population.

BACKGROUND AND OBJECTIVES: In the context of a dramatic increase in US cesarean delivery rates over the past 30 years and explicit national goals to decrease the cesarean rate, previous retrospective studies have shown that pregnant women cared for by family physicians may be less likely to undergo cesarean delivery, compared with patients cared for by obstetricians. METHODS: We conducted a retrospective chart review of 3,560 deliveries from the family practice service of a community-based family practice residency from 1986-1995, focusing primarily on cesarean delivery rates during two periods of time. During period 1 (n = 1,063), all attending were private practice obstetricians. After a transition period, all births were attended by family medicine faculty (period 2, n = 1,346). RESULTS: The total cesarean delivery rate declined from 16.7% in period 1 to 11.1% in period 2. Repeat cesareans declined from 8.5% to 2.9%. CONCLUSIONS: In this community-based residency, a change in the specialty of the attending physician was associated with a 34% decline in the cesarean delivery rate. The observed decline in the cesarean rate could not be accounted for by any change in patient demographics or secular trends in cesarean delivery rates.

207Pb-1H two-dimensional NMR spectroscopy: a useful new tool for probing lead(II) coordination chemistry.

Despite the fact that lead poisoning is the most common disease of environmental origin in the United States, the spectroscopic properties of aqueous Pb(II) coordination compounds have not been extensively investigated. Spectroscopic techniques that can be used to probe the fundamental coordination chemistry of Pb(II) will aid in both the development of water-soluble ligands that bind lead both tightly and selectively and the characterization of potential biological targets. Here, we report the preparation and characterization of a series of Pb(II) complexes of amido- derivatives of EDTA. The 207Pb chemical shift observed in these complexes (2441, 2189, and 1764 ppm for [Pb(EDTA)]2-, Pb(EDTA-N2), and [Pb(EDTA-N4)]2+, respectively) provides an extremely sensitive measure of the local environment and the charge on each complex. These shifts help to map out the lead chemical shift range that can be expected for biologically relevant sites. In addition, we report the first two-dimensional 207Pb-1H heteronuclear multiple-quantum correlation (HMQC) nuclear magnetic resonance spectra and demonstrate that this experiment can provide useful information about the lead coordination environment in aqueous Pb(II) complexes. Because this technique allows 207Pb-1H couplings through three bonds to be identified readily, 207Pb-1H NMR spectroscopy should prove useful for the investigation of Pb(II) in more complex systems (e.g., biological and environmental samples).

Maternal tobacco smoking and changes in amino acid uptake by human placental villi: induction of uptake systems, gammaglutamyltranspeptidase and membrane fluidity.

Maternal smoking depressed the active uptake of amino acids by human placentae and lowered their levels in the placenta and umbilical vein. During starvation of cells for amino acids, more amino acid carriers are induced and incorporated into the plasma membrane. A question arises whether there could be similar changes due to maternal smoking in the placental amino acid uptake carrier systems. Therefore, the characteristics of (a) the uptake of 2-amino[I-14C]-isobutyric acid (AIB) by isolated placental villi, (b) gammaglutamyltranspeptidase (GGTP), a critical enzyme of the gammaglutamyl cycle (GGC) for the uptake of amino acids in human placenta, and (c) lipid structural parameters (reciprocal of fluidity), by steady state fluorescence polarization of plasma membrane vesicles of microvilli (MV) and microsomal membranes (MM) of umbilical and chorionic plate arteries of placentae of smoking and non-smoking mothers were investigated. The above investigations gave the following results: (a) Washed placental villi of smokers exhibited higher capacity for AIB uptake than those of non-smokers. The higher uptake capacity was mainly due to increase in Vmax for AIB uptake in smokers. Km increased for placental AIB uptake in smokers. (b) Maternal smoking lowered GGTP activity of MV by decreasing its Vmax. Therefore, maternal smoking decreases the formation of gammaglutamyl-amino acid (GGAA) on the surface of trophoblast which are absorbed by the trophoblast. The degree of absorption of GGAA is considered as an inverse environmental signal for the cell to regulate amino acid transport systems. Maternal smoking seems to decrease the formation and absorption of GGAA and thereby induce the formation of new carriers for AIB uptake. (c) Maternal smoking increased the values for lipid structural order parameters and microviscosity of MV and induced tolerance against fluidization by ethyl alcohol in MM of umbilical and chorionic arteries. The alterations could increase Km for AIB uptake system and decrease the sensitivity of umbilical and chorionic arteries to vasoconstrictive substances like 5-hydroxytryptamine and catecholamine which are released by nicotine. All these changes tend to overcome the deficits produced in placental amino acid transport and satisfy the demands of the growing fetus for amino acids.

Maternal tobacco smoking and alterations in amino acid transport in human placenta: induction of transport systems.

The uptake of 14C-alpha-aminoisobutyric acid (AIB) by human placental villi from smokers and nonsmokers was measured. Washed placental villi of smokers exhibited higher uptake capacity for AIB than those of nonsmokers. The higher uptake capacity in smokers was due to increase in Vmax. Smoking increased Km and decreased affinity to AIB. This is possibly due to decreased fluidity of the plasma membrane of the villus during maternal smoking. These observations suggest that placenta utilizes its ability to increase the accumulation of amino acids during the adverse environmental conditions produced by maternal smoking.

Activities of 5-methylfurfuryltrimethylammonium iodide and related compounds at vascular endothelial muscarinic receptors of the rat aorta.

5-Methylfurfuryltrimethylammonium iodide (5-MFT) is a furan analog of muscarine and was studied for its cholinergic activity at vascular endothelial receptors of the rat aorta. Other related compounds have different substitutions at position 5 of the furan ring and include the following compounds: 5-hydroxymethyl- (5-HMFT), 5-chloromethyl- (5-CMFT), 5-bromomethyl- (5-BMFT), 5-iodomethyl- (5-IMFT), and 5-methoxy- (5-MOFT) furfuryltrimethylammonium salts. The furan analogs relaxed helical strips of rat aorta which contracted with norepinephrine (10(-6)M). These relaxations were endothelial cell-dependent. The ED50's for muscarinic activities increased in the following order: 5-MFT = ACh less than dl-muscarine less than 5-HMFT = 5-CMFT less than 5-MOFT less than 5-BMFT less than 5-IMFT. Among the furan analogs, 5-MFT was found to be a full agonist at the endothelial cells; other furan analogs were only partial agonists. The affinities and relative intrinsic efficacies of the most potent analogs decreased in the following order: ACh = 5-MFT greater than dl-muscarine greater than 5-HMFT greater than 5-CMFT. Atropine and scopolamine antagonized relaxations by furan analogs. KB values for atropine and scopolamine against ACh, 5-MFT or 5-HMFT as agonist were not different, indicating that these agonists and antagonists were acting at the same muscarinic receptors. The KB of atropine and of scopolamine increased when 5-CMFT was used as an agonist, indicating that 5-CMFT may cause relaxation by acting at other sites besides endothelial muscarinic receptors. The endothelial muscarinic receptor might be classified tentatively as of M2 or Ms type. These studies did not exclude the possible heterogeneity of the endothelial muscarinic receptors.

Opioid receptors of human placental villi modulate acetylcholine release.

The human placental villous tissue contains components of the cholinergic system and opioid receptors of the kappa type. In vitro stimulation of the villous tissue releases acetylcholine in organ baths. A selective kappa agonist, ethylketocyclazocine, inhibits the release of acetylcholine. This inhibition is reversed by the antagonist Mr 2266. The antagonist alone stimulates the release of acetylcholine 18-fold over control. These results demonstrate an interaction between the placental opioid receptors and the cholinergic system in a non-neural tissue. The modulation of acetylcholine release by endogenous opioid peptides could be one of the in vivo functions of placental opioid receptors.

Action of agonists and antagonists on adrenergic receptors in isolated porcine coronary arteries.

The adrenergic receptors of porcine coronary arteries were investigated in helically cut strips of small (less than or equal to 0.5 mm outer-diameter (od), medium (0.8-1.2 mm od), large (1.5-2.5 mm od), and very large (greater than 4 mm od) coronary arteries. Both the beta1 agonist dobutamine and the beta2 agonist terbutaline relaxed coronary arteries partially contracted by 25 mM of KCl. Dobutamine contracted small coronary arteries at 10(-5) M concentrations, then relaxed them at 10(-4) M. The beta1-adrenoceptor antagonist metoprolol contracted coronary arteries relaxed by either dobutamine or terbutaline, but the beta2 antagonist H35/25 did so only in high and probably nonselective concentrations. Alpha1-adrenoreceptor stimulating concentrations of phenylephrine did not contract any of the arteries. Metoprolol and high concentrations of H35/25 further contracted large coronary arteries partially contracted by 25 mM potassium. These contractions were blocked by verapamil and papaverine but not by atropine, phentolamine, yohimbine, mepyramine, or methysergide. This seems to indicate that beta-adrenergic receptors in porcine coronary arteries are beta1-receptors, or closely resemble beta1-receptors. They differ from many other beta1-receptors, however, in that they are stimulated by terbutaline. Alpha1 adrenoreceptors seem not to be present in these porcine coronary arteries to a significant extent. Metoprolol and high concentrations of H35/25 have a direct contractile effect in large porcine coronary artery that is not mediated by alpha-adrenergic, muscarinic, histaminergic, or serotonergic receptors but requires verapamil-sensitive calcium.

Lack of an effect of drugs acting at histamine receptors on norepinephrine-induced relaxation of porcine coronary arteries.

The effects of selective histamine receptor stimulation on relaxation to norepinephrine (NE) were determined in four sizes of spirally cut porcine coronary arteries. The four sizes of porcine coronary artery were designated very large (greater than 4.0 mm o.d.), large (1.5-2.5 mm o.d.), medium (0.8-1.2 mm o.d.), and small (less than or equal to 0.5 mm o.d.). Histamine, dimaprit, 2-pyridylethylamine, and histamine plus either mepyramine or metiamide did not alter the concentration-response curve to NE. Thus histamine receptor stimulation does not change relaxations of porcine coronary arteries to NE.

Dibenamine enhancement of histamine-induced relaxation of the rabbit mesenteric artery.

Helically cut strips of rabbit mesenteric artery relax when exposed to histamine if their histamine H1 receptors are first blocked by 7 X 10(-6) M mepyramine. Relaxations are potentiated by 20 min pretreatment with 10(-6) M dibenamine. This dibenamine regimen also enhances relaxation of the strips to the selective H2 receptor agonist dimaprit, and to a lesser extent to papaverine which does not act on histamine receptors. This enhancement occurs both at 38 degrees and 22 degrees, and in mesenteric artery strips from rabbits reserpinized to deplete amine stores. Histamine has a greater relaxant effect on mesenteric artery strips at 22 degrees than at 38 degrees, normally. Dibenamine-treated strips do not relax more at the lower temperature, however. Thus, dibenamine nonselectively enhances relaxations of mesenteric artery and may enhance histamine-induced relaxations by an additional mechanism.

Effects of dithiothreitol on the isolated rabbit mesenteric artery.

Rabbit mesenteric artery strips exposed to 10(-3) M dithiothreitol (DTT) were contracted with a series of concentrations of histamine and 2-pyridylethylamine (PEA). DTT exposure increased the sensitivity to histamine 100-fold but increased the sensitivity to PEA only 4-fold. DTT did not reduce dimaprit-induced relaxations, but reduced histamine-induced relaxations. Following a high concentration of histamine (10(-3) M), DTT itself produced a sustained, slowly developing contraction (29 +/- 6.8% of the maximal contraction) relaxed by 7 X 10(-6) M mepyramine but not by 10(-6) M phentolamine. Metiamide (3 x 10(-5) M) potentiated DTT-induced contractions (29 +/- 6.8 before, 57 +/- 7.5% after metiamide, as a percent of maximal contraction). Changing the bathing fluid and repeating DTT exposure slowly relaxed previously contracted strips. DTT did not prevent the increase in sensitivity of relaxant histamine receptors on exposure to cold. We conclude that DTT, in addition to potentiating histamine H1-receptor responses, releases histamine presumably from non-mast cell pools when they are loaded with a high concentration of exogenous histamine.