Studies on a 50S Ribosomal Precursor Particle as a Substrate for \u3cem\u3eerm \u3c/em\u3eE Methyltransferase Enzyme in \u3cem\u3eStaphylococcus aureus \u3c/em\u3e.

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. We are interested in investigating the true substrate for methylation because it is known from our work and the work of others that fully assembled 50S subunits are not substrates for methylation. We have published a model for 50S ribosomal subunit formation where, the precursor particle that accumulates in erythromycin treated cells is a target for methyl transferase activity. Current studies are aimed at investigating the role of the precursor particle as substrate for ermE methyltransferase activity and the competition between this enzyme and erythromycin for the 50S precursor particle. Slot-blot hybridization experiments have identified the presence of 23S rRNA in the 50S precursor region. Quantitation of the 23S rRNA in these blots also revealed that the percentage of the precursor increased as the concentration of erythromycin was increased in the growth media. Ribosomal proteins of S. aureus were studied by two-dimensional gel electrophoresis. Protein content of the 50S precursor particle was analyzed by MALDI-TOF. These studies have identified 16 50S ribosomal proteins in the precursor region. Methyltransferase assays showed that 50S precursor particle was a substrate for ermE methyltransferase. Importantly, RNA that is already assembled into 50S subunits was not a substrate for the enzyme. Inhibition curves showed that macrolide, lincosamide, and streptogramin B (MLSB) drugs bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. Competition experiments suggested that the enzyme can displace erythromycin from the 50S precursor particle and that erm methyltransferase has a lower association constant for the precursor particle compared to that of the erythromycin. This suggests that higher concentrations of erythromycin are needed to combat erm induced resistance. These studies shed light on the interaction of ermE methyltransferase and erythromycin in the clinically important pathogen S. aureus

Grape Powder Improves Age-Related Decline in Mitochondrial and Kidney Functions in Fischer 344 Rats

We examined the effects and mechanism of grape powder- (GP-) mediated improvement, if any, on aging kidney function. Adult (3-month) and aged (21-month) Fischer 344 rats were treated without (controls) and with GP (1.5% in drinking water) and kidney parameters were measured. Control aged rats showed higher levels of proteinuria and urinary kidney injury molecule-1 (KIM-1), which decreased with GP treatment in these rats. Renal protein carbonyls (protein oxidation) and gp91phox-NADPH oxidase levels were high in control aged rats, suggesting oxidative stress burden in these rats. GP treatment in aged rats restored these parameters to the levels of adult rats. Moreover, glomerular filtration rate and sodium excretion were low in control aged rats suggesting compromised kidney function, which improved with GP treatment in aged rats. Interestingly, low renal mitochondrial respiration and ATP levels in control aged rats were associated with reduced levels of mitochondrial biogenesis marker MtTFA. Also, Nrf2 proteins levels were reduced in control aged rats. GP treatment increased levels of MtTFA and Nrf2 in aged rats. These results suggest that GP by potentially regulating Nrf2 improves aging mitochondrial and kidney functions

Characteristics of a 50S Ribosomal Subunit Precursor Particle as a Substrate for ErmE Methyltransferase Activity and Erythromycin Binding in Staphylococcus Aureus

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. A model for 50S ribosomal subunit formation suggests that the precursor particle which accumulates in erythromycin treated cells is the target for methyl transferase activity. Hybridization experiments identified the presence of 23S rRNA in the 50S precursor particle. The protein content of the 50S precursor particle was analyzed by MALDI-TOF mass spectrophotometry. These studies have identified 23 of 36 50S ribosomal proteins in the precursor. Methyltransferase assays demonstrated that the 50S precursor particle was a substrate for ermE methyltransferase. Competition experiments indicated that the enzyme could displace erythromycin from the 50S precursor particle and that the methyltransferase had a higher association constant for the precursor particle compared to that of erythromycin. Inhibition experiments showed that macrolide, lincosamide and streptogramin B compounds bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. These studies shed light on the interaction of ermE methyltransferase and erythromycin in this clinically important pathogen

Simulated vehicle exhaust exposure (SVEE) in rats impairs renal mitochondrial function

Purpose  Vehicle exhaust emissions primarily comprise of nitrogen, oxygen, water, CO2, NO2, CO, hydrocarbons and particulate matter. While adverse effects of hydrocarbon and particulate matter on cardiovascular functions are known, the effect of pro-oxidants CO2, NO2 and CO are not clear. Methods  Here, using an animal model of a simulated mixture of pro-oxidants (0.04% CO2, 0.9 ppm NO2 and 3 ppm CO with air as a base), we examined the effect of simulated vehicle exhaust exposure (SVEE) on various cardiovascular parameters. Male Sprague-Dawley rats were exposed to SVEE or ambient air (Control: CON) for 30 min/day for 2 weeks. Thereafter, systolic and diastolic blood pressure, heart rate and glomerular filtration rate were measured. Later, rats were sacrificed, blood plasma and kidneys were collected. Results  The systolic and diastolic blood pressure, heart rate and glomerular filtration rate remained unchanged. Plasma corticosterone increased in SVEE rats when compared to CON group. Plasma 8-isoprostane, a systemic marker of oxidative stress, increased while total antioxidant capacity decreased in SVEE but not in CON. Kidney cortical tissue homogenates exhibited increase in superoxide, hydrogen peroxide and protein carbonylation in SVEE but not CON, all indicative of heightened oxidative stress. Renal cortical mitochondrial SOD activity was significantly reduced in SVEE than CON. Conclusion  Significant decline in mitochondrial respiration and oxygen consumption was observed, in addition to low ATP, reduced ATP synthase and cytochrome C oxidase levels, as well as accelerated mitochondrial fission, and reduced fusion processes, were observed in SVEE than CON rats, all indicative of renal mitochondrial impairment

Agonist induced β₂AR signaling is required for mucin production in response to IL-13 in NHBE cells.

<p>NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 10 μM nadolol (a non-selective inverse agonist of βARs) or 10 μM alprenolol (a non-selective β₂AR blocker with no inverse agonist activity) for 14 days. <b>A</b>: MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. <b>B</b>: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s004" target="_blank">S4A Fig</a>). <b>C</b>: Quantification of intracellular mucin glycoproteins in response to different ligands. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s004" target="_blank">S4B Fig</a>). Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to + epinephrine and + epinephrine + IL-13 treated cells respectively.</p

Epinephrine is required for mucin production in response to IL-13 in NHBE cells.

<p><b>A</b>: NHBE cells were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were treated with 20 ng/ml IL-13 for 14 days, total RNA was harvested and then MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to the corresponding treatment control (in the absence of IL13). <b>B</b>: Representative images of immunofluorescence with a rabbit antibody against human mucin 5AC (red) (scale bar = 100 μm). The Transwell membranes were incubated with DAPI to counterstain the nuclei (blue). Incubation with antibody diluent showed no red fluorescence (data not shown). The ratio of integrated fluorescence density of each group to the integrated mucin 5AC density of the corresponding control group was calculated and expressed as fold change. <b>C</b>: PAFS staining of NHBE cells to quantify total intracellular mucin glycoproteins. Representative images are shown. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to the corresponding control cells (in the absence of IL-13 treatment). Data are presented as means ± SEM from three donors. *, † and ¥ indicate p<0.05 significance as compared to + epinephrine,−epinephrine and −epinephrine + IL-13 treated cells respectively.</p