Azithromycin and Clarithromycin Inhibition of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

The ID50 values for azithromycin and clarithromycin inhibition of translation and of 50S assembly in Staphylococcus aureus cells have been measured. For clarithromycin, 50% inhibition of growth occurred at 0.075 μg/ml, and the effects on translation and 50S formation were equivalent at 0.15 μg/ml. The inhibition of these processes by azithromycin was less effective, with an ID50 of 2.5 μg/ml for growth and 5 μg/ml for inhibition of translation and 50S formation. The additive effects of each of these drugs on translation and 50S formation account quantitatively for their observed influence on cellular growth rates. In macrolide-treated cells, there was also a direct relationship between the loss of ribosomal RNA from the 50S subunit and its accumulation as oligoribonucleotides. These results are compared with the previously described effects of erythromycin on these same processes

50S Ribosomal Subunit Synthesis and Translation Are Equivalent Targets for Erythromycin Inhibition in Staphylococcus aureus

Macrolide antibiotics like erythromycin can prevent the formation of the 50S ribosomal subunit in growing bacterial cells, in addition to their inhibitory effect on translation. The significance of this novel finding has been further investigated. The 50% inhibitory doses of erythromycin for the inhibition of translation of 50S subunit assembly in Staphylococcus aureus cells were measured and were found to be identical. Together they account quantitatively for the observed effects of erythromycin on cell growth rates. There is also a direct relationship between the loss of rRNA from the 50S subunit and its accumulation as oligoribonucleotides in cells. The importance of this second site for erythromycin inhibition of bacterial cell growth is discussed

Macrolide Antibiotic Inhibition of Translation and 50S Ribosomal Subunit Assembly in Methicillin-Resistant Staphylococcus aureus Cells

Methicillin-resistant Staphylococcus aureus cells were treated with three macrolide antibiotics to examine the inhibitory effect of the drugs on the growth rate and cell viability. Inhibition of protein synthesis and 50S ribosomal subunit assembly were also examined. The growth rate and cell viability were reduced by each antibiotic in both erythromycin-susceptible and erythromycin-resistant MRSA organisms. Translation and the formation of the 50S ribosomal subunit were inhibited to an equal extent in the erythromycin-susceptible cells, but protein synthesis was affected to a greater extent by each macrolide in the erythromycin-resistant organisms. Clarithromycin was the most inhibitory of the three compounds, followed by erythromycin and azithromycin in relative effectiveness. The use of these compounds against MRSA organisms is discussed

Inhibition of Phorbol Ester-Stimulated Arachidonic Acid Release by Alkylglycerols

Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C

A Comparison of the Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus Aureus Cells by Nine Different Macrolide Antibiotics

Nine structurally similar macrolide antibiotics were tested at a concentration of 0.5 μg/ml for their relative inhibitory effects on ribosome functions in Staphylococcus aureus cells. Eight of the compounds examined inhibited protein synthesis at this concentration. Seven of the nine compounds were also effective in blocking formation of the 50S ribosomal subunit. Roxithromycin and 14-hydroxy clarithromycin inhibited protein synthesis to a greater extent than they affected 50S subunit formation. Conversely, the compound 11,12-carbonate-3 deoxy-clarithromycin affected 50S assembly more than translation. Only clarithromycin had any effect on 30S ribosomal subunit assembly. The decline in growth rate and cell number was proportional to the effect on ribosome formation or function by each compound. These inhibitory activities can be related to structural differences between these macrolide antibiotics

Influence of Constitutive Model Parameters on the Predicted Migration of DNAPL in Heterogeneous Porous Media

This study examines the influence of constitutive models and their parameters on predictions of the spatial and temporal distribution of a finite release of a dense, nonaqueous phase liquid (DNAPL) into a two-dimensional, spatially correlated random permeability field. The base case simulation employed a comprehensive constitutive model that was validated against relevant one-dimensional laboratory experiments. The base case was perturbed in the course of nine individual simulations, where each simulation examined the consequences of simplifying a single model characteristic. None of the nine subsequent simulations was able to reproduce, within ±10%, the spatial and temporal migration characteristics of the nonwetting fluid body at late time predicted by the base case. Capillary pressure-saturation relationships that do not incorporate specific displacement and terminal pressures are demonstrated to severely overpredict the spatial extent of nonwetting fluid advancement. This suggests that van Genuchten-based models may not be suitable for predicting DNAPL migration in saturated porous media. Not accounting for any one of hysteresis, nonwetting phase trapping, or the proper curvature or end-point values of the nonwetting phase imbibition relative permeability curve profoundly influenced the time predicted for all nonwetting fluid movement to cease. The practical implication of this study is that an appropriate, comprehensive constitutive model, characterized with suitable parameter values, is necessary to accurately simulate a complete DNAPL release below the water table in both space and time

Abrogation of MAP4K4 protein function causes congenital anomalies in humans and zebrafish.

We report 21 families displaying neurodevelopmental differences and multiple congenital anomalies while bearing a series of rare variants in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4). MAP4K4 has been implicated in many signaling pathways including c-Jun N-terminal and RAS kinases and is currently under investigation as a druggable target for multiple disorders. Using several zebrafish models, we demonstrate that these human variants are either loss-of-function or dominant-negative alleles and show that decreasing Map4k4 activity causes developmental defects. Furthermore, MAP4K4 can restrain hyperactive RAS signaling in early embryonic stages. Together, our data demonstrate that MAP4K4 negatively regulates RAS signaling in the early embryo and that variants identified in affected humans abrogate its function, establishing MAP4K4 as a causal locus for individuals with syndromic neurodevelopmental differences