A number of different antibiotics that prevent translation by binding to the 50S ribosomal subunit of bacterial cells have recently been shown to also prevent assembly of this subunit. Antibacterial agents affecting 30S particle activities have not been examined extensively for effects on small subunit formation. The aminoglycoside antibiotics paromomycin and neomycin bind specifically to the 30S ribosomal subunit and inhibit translation. These drugs were examined in Staphylococcus aureus cells to see whether they had a second inhibitory effect on 30S particle assembly. A 3H-uridine pulse and chase assay was used to examine the kinetics of subunit synthesis in the presence and absence of each antibiotic. 30S subunit formation was inhibited by both compounds. At 3 μg/mL each antibiotic reduced the rate of 30S formation by 80% compared with control cells. Both antibiotics showed a concentration-dependent inhibition of particle formation, with a lesser effect on 50S particle formation. For neomycin, the IC50 for 30S particle formation was equal to the IC50 for inhibition of translation. Both antibiotics reduced the viable cell number with an IC50 of 2 μg/mL. They also inhibited protein synthesis in the cells with different IC50 values (2.5 and 1.25 μg/mL). This is the second demonstration of 30S ribosomal subunit-specific antibiotics that prevent assembly of the small subunit.

Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with 3H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug.

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC50 values of 1.5 μg/ml. Protein synthesis was inhibited in cells with a similar IC50 concentration (1.25 μg/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism.

No description available.

A substantial number of antimicrobial agents target some activity of the bacterial ribosome for inhibition. Mechanistic studies and recent structural investigations of the ribosome have identified the binding sites and presumed mechanism of inhibitory activity for some compounds. A second target for many of these antibiotics has recently been examined. Formation of both 30S and 50S ribosomal subunits in bacterial cells is impaired by translational inhibitors. For many antimicrobial agents, inhibition of this target is equivalent to inhibition of translation in preventing cell growth. This review will describe features of this new target including the types of compounds which affect particle assembly and differences in the process in different microorganisms. The characteristics of this new target will be identified and aspects of a model to explain this new inhibitory activity will be explored.

The aims of the study were to determine how aggregates of intrinsic cardiac (IC) neurons transduce the cardiovascular milieu versus responding to changes in central neuronal drive and to determine IC network interactions subsequent to induced neural imbalances in the genesis of atrial fibrillation (AF). Activity from multiple IC neurons in the right atrial ganglionated plexus was recorded in eight anaesthetized canines using a 16-channel linear microelectrode array. Induced changes in IC neuronal activity were evaluated in response to: (1) focal cardiac mechanical distortion; (2) electrical activation of cervical vagi or stellate ganglia; (3) occlusion of the inferior vena cava or thoracic aorta; (4) transient ventricular ischaemia, and (5) neurally induced AF. Low level activity (ranging from 0 to 2.7 Hz) generated by 92 neurons was identified in basal states, activities that displayed functional interconnectivity. The majority (56%) of IC neurons so identified received indirect central inputs (vagus alone: 25%; stellate ganglion alone: 27%; both: 48%). Fifty per cent transduced the cardiac milieu responding to multimodal stressors applied to the great vessels or heart. Fifty per cent of IC neurons exhibited cardiac cycle periodicity, with activity occurring primarily in late diastole into isovolumetric contraction. Cardiac-related activity in IC neurons was primarily related to direct cardiac mechano-sensory inputs and indirect autonomic efferent inputs. In response to mediastinal nerve stimulation, most IC neurons became excessively activated; such network behaviour preceded and persisted throughout AF. It was concluded that stochastic interactions occur among IC local circuit neuronal populations in the control of regional cardiac function. Modulation of IC local circuit neuronal recruitment may represent a novel approach for the treatment of cardiac disease, including atrial arrhythmias.

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5′-CTCTCG1G 2CG3CCATCAC-3′): 5′-CG1* G2*CG3CC-3′, 5′-CG1G 2*CG3*CC-3′, and 5′-CG 1*G2CG3*CC-3′ (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G 3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

The influence of stress agents, whether social, restraint, malnutrition or mild unpredictable, during the fetal-prenatal, infant-postnatal, adolescent or young adult phases of the lifespan generally, but not always, implies disruption of the normal process of development. Several notions of stress including the adaptive calibration model, adaptive emotional processes and arousability, GABAergic integrity and nutrient deficiency, and resilience influence the physiological and behavioural expressions of maternal stress, affecting nursing behaviour and offspring outcome. The adaptive/maladaptive effects of stress in humans are affected by developmental programming of the hypothalamic-pituitary-adrenal (HPA) axis and other neuroendocrine systems related to stress that may facilitate expressions of resilience. The adaptive/maladaptive effects of stress in animal models outline dysfunctional HPA axis and brain regional alterations of phenotypic expressions that interact with epigenetic mechanisms and developmental plasticity. Maladaptive stress regulation in adolescence is influenced by several factors, not the least being serotonergic, glucocorticoid and regional integrity pertaining to trauma in adolescence. The occurrence of oxidative stress may imply damage but the propensity for hormesis, a notion not unrelated to resilience, provides opportunities for long-lasting health benefits.

Recent multichannel electrode array technology has enabled the simultaneous recording of multiple cardiac neurons located in ganglia on a beating heart. These new bioelectric signals are contaminated by the electrical activity of the atrial muscle just underneath. These atrial waveforms may mask relevant neuronal activity. In this paper, we evaluate the application of a principal component analysis technique to suppress atrial activity (AA) and reveal hidden neuronal activity. Neuronal signals were recorded in situ using a 16-channel electrode in an open-chest, anesthetized dog in sinus rhythm. Validation of AA cancellation was performed by comparing neuron spike waveforms extracted from within AA with those found in AA-free time intervals. Results showed that consistent neuronal waveforms can be identified within AA in order to improve the detection of neuron firings.

The purpose of this letter is to expand a discussion, published recently in Molecular Immunology, on the post-secretion gain of function by immunoglobulins. It was reported that some circulating IgG molecules in all healthy individuals acquire novel antigen-binding specificity after exposure to conditions that change protein conformation, such as acidic pH. Another protein, C-reactive protein, also acquires novel ligand-binding specificity post-secretion after a switch from its native pentameric conformation to non-native pentameric conformation. Thus, that the functions of a protein depend upon its alternate structural states, and therefore on the surrounding milieu, is a more general phenomenon for some ancient molecules of the immune system than previously thought.