Characterization of the ribosomal RNA operons of Haloarcula marismortui

The genome of Haloarcula marismortui contains two ribosomal RNA operons, designated as rrnA and rrnB (and possibly a third operon designated as rrnC) of which the characterization of the rrnA and rrnB operons are presented. Characterization of the rrnA and rrnB operons involved the analysis of primary and secondary structures and in vivo studies of the primary transcripts and processing intermediates. It was found that the gene orders of the rrnA and rrnB operons were 5-16S rRNA-tRNAA l a-23S rRNA-5S rRNA-tRNACys- 3' and 5-16S rRNA-23S rRNA-5SrRNA-3', respectively. Computing the substitution rates for the entire rrnA and rrnB operons demonstrated that the major differences are localized in the non coding regions, that is the regions including the 5'-flanking of the 16S rRNA, 16S-23S rRNA spacer and the 3'-flanking of the 5S rRNA gene. The percentage similarities between the 16S, 23S and 5S rRNAs of rrnA and rrnB are 95%, 98.7% and 98.3%, respectively. A pairwise sequence comparison between the 23S rRNA sequence of the rrnC operon (Brombach et al., 1989) and the other two operons, rrnA and rrnB, revealed that the sequence similarities are 98.8% and 99.6%, respectively. The 5S rRNA sequence from the rrnC operon is identical to the rrnA sequence. The nucleotide substitutions within the 16S rRNA genes of rrnA and rrnB operons are concentrated in three separate domains 58-321, 508-823 and 986-1158. About 60% of the substitutions are concentrated within the 508-823 domain and are compensatory, affecting both components of the nucleotide base pairs within defined rRNA helices. Using nuclease Sl protection assays, it was shown that the 16S rRNAs from the rrnA and rrnB operons are expressed and present in intact 70S ribosomes. A comparison of the 23S rRNAs from the rrnA and rrnB operons showed that the substitutions are located within the variable regions of domains I, m, IV and VI of the universal secondary structures of 23S rRNAs. The 5S rRNA sequences of the two operons differ at two nucleotide positions in the helix IV of the universal secondary structure for the 5S rRNA. The 5'-flarucing regions of the rrnA contains four tandem promoters whereas the rrnB operon contains a single tandem promoter and a second promoter-like sequence. An internal promoter sequence was present within the 16S-23S spacer regions of all three operons from Ha. marismortui. Putative secondary structures of the primary transcripts from the rrnA operon showed that the 16S and 23S rRNAs are surrounded by inverted repeat structures containing the "bulge-helix-bulge" motif which is recognized by a processing endonuclease. In the case of the rrnB operon, the inverted repeat structure surrounding the 23S rRNA is identical to that of the rrnA operon and processing follows the same pathway. However, the inverted repeat structure surrounding 16S rRNA from rrnB does not contains the "bulge-helix- bulge" motif and its processing follows a distinct pathway. The 16S rRNA processing of the rrnB occurs at a single position within the 5'-flanking region and at three positions within the 16S-23S spacer region. The nucleotides present in these cleavage sites and their surrounding regions showed no sequence conservation. The 5S rRNA processing occurs close to or at its 5'- and 3'-ends. Two apparent termination sites were detected for the rrnA operon and they were found to be located downstream of the tRNACys RNA. Phylogenetic analysis of the 508-823 region of the 16S rRNA, the entire 16S rRNA, 23S rRNA genes and the combination of 16S-23S-5S rRNA genes were performed using the PAUP program. The phylograms from the 16S rRNA, 23S rRNA and the 16S-23S-5S rRNA sequences indicated that the rrnA and rrnB group together and the rate of divergence of the rrnB operon is higher than that of the rrnA operon. However, comparison of the 508- 823 regions showed that the two operons do not group together and that rrnB is evolving slower than rrnA. Based on the comparisons made between the rrnA and rrnB operons, it is obvious that the rrnB operon is different from the rrnA operon in its gene order, rRNA sequences and 16S rRNA processing and also probably evolving at a different rate than the rrnA operon.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

In Vivo Neocortical [K]o Modulation by Targeted Stimulation of Astrocytes

A normally functioning nervous system requires normal extracellular potassium ion concentration ([K]o). Throughout the nervous system, several processes, including those of an astrocytic nature, are involved in [K]o regulation. In this study we investigated the effect of astrocytic photostimulation on [K]o. We hypothesized that in vivo photostimulation of eNpHR-expressing astrocytes leads to a decreased [K]o. Using optogenetic and electrophysiological techniques we showed that stimulation of eNpHR-expressing astrocytes resulted in a significantly decreased resting [K]o and evoked K responses. The amplitude of the concomitant spreading depolarization-like events also decreased. Our results imply that astrocytic membrane potential modification could be a potential tool for adjusting the [K]o

Severe Hypoglycemia in a Juvenile Diabetic Rat Model: Presence and Severity of Seizures Are Associated with Mortality

<div><p>It is well accepted that insulin-induced hypoglycemia can result in seizures. However, the effects of the seizures, as well as possible treatment strategies, have yet to be elucidated, particularly in juvenile or insulin-dependent diabetes mellitus (IDDM). Here we establish a model of diabetes in young rats, to examine the consequences of severe hypoglycemia in this age group; particularly seizures and mortality. Diabetes was induced in post-weaned 22-day-old Sprague-Dawley rats by streptozotocin (STZ) administered intraperitoneally (IP). Insulin IP (15 U/kg), in rats fasted (14–16 hours), induced hypoglycemia, defined as <3.5 mM blood glucose (BG), in 68% of diabetic (STZ) and 86% of control rats (CON). Seizures occurred in 86% of STZ and all CON rats that reached hypoglycemic levels with mortality only occurring post-seizure. The fasting BG levels were significantly higher in STZ (12.4±1.3 mM) than in CON rodents (6.3±0.3 mM), resulting in earlier onset of hypoglycemia and seizures in the CON group. However, the BG at seizure onset was statistically similar between STZ (1.8±0.2 mM) and CON animals (1.6±0.1 mM) as well as between those that survived (S+S) and those that died (S+M) post-seizure. Despite this, the S+M group underwent a significantly greater number of seizure events than the S+S group. 25% glucose administered at seizure onset and repeated with recurrent seizures was not sufficient to mitigate these continued convulsions. Combining glucose with diazepam and phenytoin significantly decreased post-treatment seizures, but not mortality. Intracranial electroencephalograms (EEGs) were recorded in 10 CON and 9 STZ animals. Predictive EEG changes were not observed in these animals that underwent seizures. Fluorojade staining revealed damaged cells in non-seizing STZ animals and in STZ and CON animals post-seizure. In summary, this model of hypoglycemia and seizures in juvenile diabetic rats provides a paradigm for further study of underlying mechanisms. Our data demonstrate that severe hypoglycemia (<2.0 mM) is a necessary precondition for seizures, and the increased frequency of these seizures is associated with mortality.</p></div

Effects of treatments (see Table 1) on the seizure scores and mortality in STZ rats.

<p><b>A:</b> Mean number of seizures in (*) <b>glu</b> rats: S+S: 1.6±0.2 (n = 17) and S+M: 4.4±1.2 (n = 5) (p<0.001); <b>ac+1xglu</b> rats: S+S: 1.3±0.2 (n = 8) and S+M: 1.4±0.2 (n = 8). (**) Significant difference also exists between <b>glu</b> rats: S+M and <b>ac+1xglu</b> rats: S+M (p<0.05) <b>B:</b> Mean maximum seizure score attained in <b>glu</b> rats: S+S: 4.3±0.4 (n = 17) and S+M: 5.5±0.4 (n = 5); (*) <b>ac+1xglu</b> rats: S+S: 4.0±0.6 (n = 8) and S+M: 6.1±0.6 (n = 8) (p<0.01).</p

Mean blood glucose level measured hourly related to treatment and survival.

<p>S+S Glu: Seizure + Survival; glucose treated.</p><p>S+S AC+1XGLU: Seizure + Survival; glucose and anticonvulsant treated.</p><p>S+M Glu: Seizure + Mortality; glucose treated.</p><p>S+M AC+1XGLU: Seizure + Mortality; glucose and anticonvulsant treated.</p

Seizure score to characterize and quantify the observed seizures.

<p>Seizure score to characterize and quantify the observed seizures.</p

Efficacy of treatment strategies (see Table 1) in preventing subsequent seizures and mortality in STZ rats.

<p><b>A</b>: BG decrease is not significantly different in seizing animals regardless of treatment; <b>glu</b>: seizure+ survival (S+S Glu), <b>ac+1xglu</b>: S+S AC, <b>glu</b>: seizure + mortality (S+M Glu), <b>ac+1xglu</b>: S+M AC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083168#pone-0083168-t004" target="_blank">Table 4</a>). B: No significant difference in the incidence of seizures post-treatment in <b>ac+1xglu</b>: 24% (n = 5/21) compared with glu: 55% (n = 12/22) (p<0.05). (*) Significantly lower incidence of seizures post-treatment in <b>ac+multiple glu</b>: 15% (n = 2/14) compared with <b>glu</b>: 55% (n = 12/22) (p<0.02) C: (*) Significantly higher survival rate in <b>ac+multiple glu</b>: 93% (n = 1/14) compared with <b>ac+1xglu</b>: 48% (n = 10/21) (p<0.02). No significant difference in survival rate between <b>ac+multiple glu</b>: 93% (n = 13/14) and <b>glu</b>: 77% (n = 17/22).</p

Comparing the association between seizure severity and mortality in CON and STZ rats.

<p><b>A:</b> No significant difference in the mean score of the first seizure treated in CON rats: S+S: 4.3±0.5 (n = 11) and S+M: 6.0±0.4 (n = 5) and STZ rats: S+S: 3.4±0.3 (n = 17) and S+M: 4.7±0.7 (n = 5) <b>B:</b> No significant difference in the mean maximum seizure score observed in CON rats: S+S: 4.9±0.4 (n = 11) and S+M: 6.5±0.3 and STZ rats: S+S: 4.3±0.4 (n = 17) and S+M: 5.5±0.4 (n = 5) <b>C:</b> A statistically significant difference in the mean number of seizures between (*) CON rats: S+S: 1.6±0.3 (n = 11) and S+M: 7.8±2.7 (n = 5; p<0.01) and between (*) STZ rats: S+S: 1.6±0.2 (n = 17) and S+M: 4.4±1.2 (n = 5; p<0.001).</p

Mean blood glucose level measured hourly related to outcome; (±S.E.M).

<p>NS: No Seizure.</p><p>S+M: Seizure + Mortality.</p><p>S+S: Seizure + Survival.</p