Comparisons between antimicrobial pharmacodynamic indices and bacterial killing as described by using the Zhi model

Various suggestions have been made for empirical pharmacodynamic indices of antibiotic effectiveness, such as areas under the drug concentration-time curve in serum (AUC), AUC > MIC, AUC/MIC, area under the inhibitory curve (AUIC), AUC above MIC, and time above MIC (T > MIC). In addition, bacterial growth and killing models, such as the Zhi model, have been developed. The goal of the present study was to compare the empirical behavior of the Zhi model of bacterial growth and killing with the other empirical pharmacodynamic indices described above by using simulated clinical data analyzed with the USC*PACK PC clinical programs for adaptive control of drug therapy, with one model describing a concentration-dependent antibiotic (tobramycin) and another describing a concentration-independent antibiotic (ticarcillin). The computed relative number of CFU was plotted against each pharmacodynamic index, with each axis parameterized over time. We assumed that a good pharmacodynamic index should present a clear and continuous relationship between the time course of its values and the time course of the bacterial killing as seen with the Zhi model. Preliminary work showed that some pharmacodynamic indices were very similar. A good sensitivity to the change in the values of the MIC was shown for AUC/MIC and also for T > MIC. In addition, the time courses of some other pharmacodynamic indices were very similar. Since AUC/MIC is easily calculated and shows more sensitivity, it appeared to be the best of the indices mentioned above for the concentration-dependent drug, because it incorporated and used the MIC the best. T > MIC appeared to be the best index for a concentration-independent drug. We also propose a new composite index, weighted AUC (WAUC), which appears to be useful for both concentration-dependent and concentration-independent drugs.     

Differential chromosome sensitivity to 5-azacytidine in Alzheimer''s disease

The chemiluminescence (CL) technique with scavengers for superoxide anion (superoxide dismutase) and hydrogen peroxide (catalase) was used to characterize the generation of reactive oxygen species (ROS) inside and outside the human neutrophil after stimulation with both soluble (formyl-methionyl-leucyl-phenylalanine, FMLP) and particulate (urate crystals, zymosan, oxidized LDL) stimuli. Depending on the stimulus used, ROS generation differed in composition and absolute amounts. The ratio between extracellularly and intracellularly produced ROS ranged from 0.3 (zymosan) to 4.2 (FMLP). While enhancing substantially FMLP-stimulated CL, horseradish peroxidase inhibited CL induced by particulate stimuli by 40-80%. Furthermore, an azide-insensitive and therefore peroxidase-independent part of CL was found in FMLP-, LDL- and zymosan-stimulated cells. The results indicate that different agonists may lead through distinct chemical pathways to neutrophil luminol-amplified light generation.     

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Sex pheromone of the oleander scale, Aspidiotus nerii: structural characterization and absolute configuration of an unusual functionalized cyclobutane

The sex pheromone emitted by the female oleander scale, Aspidiotus nerii (Homoptera, Diaspididae), has been isolated and characterized as (1R, 2S)-cis-2-isopropenyl-1-(4'-methyl-4'-penten-1'-yl)cyclobutaneethanol acetate by using advanced MS and NMR spectroscopic methods, as well as a variety of microderivatization sequences. The structure has been confirmed by stereo- and enantioselective synthesis of the four possible stereoisomers. The absolute configuration has been determined by comparison of the activity of the cis (1S,2R) and (1R, 2S) enantiomers with that exhibited by the natural material in greenhouse bioassays and field tests. The structure of this sesquiterpenoid pheromone is new in the coccids and in the pheromone field in general.     

The dual coated pit pathway hypothesis: vertebrate cells have both ancient and modern coated pit pathways for receptor mediated endocytosis

The SimPlate Total Plate Count (TPC) test, developed by IDEXX Laboratories, Inc., detects and quantitates total bacterial concentration in food after 24 h of incubation. The performance of SimPlate TPC was compared with that of the plate count agar (PCA) method for enumerating total bacterial concentration of 255 food samples representing 15 different food matrixes. Total bacterial counts on SimPlate TPC were measured after 24 h of incubation and plotted against values obtained from PCA after 48 h. Simple regression analysis of the data showed strong correlation between the methods (r = 0.95); the sensitivity of SimPlate TPC for foodborne bacteria was 96% relative to PCA (slope = 0.96). It was concluded that SimPlate TPC is a suitable alternative for the detection and quantitation of foodborne bacteria. The method has been granted Performance Tested Certification by the AOAC Research Institute.     

A conserved TN8TCCT motif in the octapeptide-encoding region of Pax genes which has the potential to direct cytosine methylation

Our previous findings have shown that the developmental genes Pax7 and Pax3 are differentially methylated; the gene region that encodes the paired domain is hypomethylated, whereas the region that encodes the homeodomain is hypermethylated. For this reason, the known DNA sequence between the paired and homeoboxes was analysed for the presence of a conserved DNA motif to which a modifying protein could bind in order to direct the methylation or demethylation of surrounding gene sequences. The octapeptide-encoding region was found to contain several nucleotides that were highly conserved throughout the Pax gene family from phylogenetically distant species. The most conserved nucleotides are thought to comprise a motif TN8TCCT where N8=any combination of eight nucleotides. A conserved octapeptide-like-encoding sequence containing the TN8TCCT motif was also found in non-Pax genes of higher eukaryotes and in the non-coding strand of plants. Moreover, differential methylation seems to be associated with the presence of the TN8TCCT motif in p53 and the human oestrogen receptor genes. The presence of the TN8TCCT motif within an octapeptide-like-encoding sequence in human T-cell leukaemia virus type 1 might suggest that the putative recognition motif may have been introduced into various host genomes via some form of retroviral agent.     

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Iq from the integron of a natural multiresistance plasmid

The nucleotide sequence of a newly identified amikacin resistance gene, aac(6')-Iq (551 bp), is reported. It has 68.4 and 94.4% homology with the aac(6')-Ia gene and the recently described aac(6')-Ip gene, respectively. Analysis of its flanking sequences indicated that it is in the first cassette of a class I integron and has an attC site (59-base element) 108 bp in length.     

High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.