Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.     

Chemoreception of an oviposition deterrent associated with eggs in pieris brassicae

Electro-antennograms (EAG) in Pieris brassicae butterflies showed graded responses to the odour of intact conspecific eggs, and also to leaf and flower volatiles. Contributing to this summated potential are at least two types of sensilla on each segment of the antenna, although the terminal 6–8 segments do not respond proportionately. Analysis of the activity profiles of single olfactory cells to these compounds shows a typical generalist spectrum, with 0–3 cells per sensillum responding to the stimulus by an increase or decrease in spike frequency. The pheromone associated with eggs and deterring other females from ovipositing is not present in ovaries, but is produced in the accessory glands. It is water-soluble, has a low volatility and causes an increase in spike frequency in tarsal contact chemoreceptors.Behavioural evidence for the presence of a three-tier discrimination (visual detection of eggs or larvae: olfactory detection of aversion pheromone during fluttering over the leaves: contact discrimination by tarsae after landing (Rothschild & Schoonhoven, 1977)) is supported by electrophysiological results, which indicate the involvement of olfactory and contact chemoreceptors in recognising the presence of eggs.

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Zusammenfassung Elektroantennogramme (EAG) von Pieris brassicae Faltern zeigten abgestufte Reaktionen auf den Geruch konspezifischer Eier und auch auf flüchtige Stoffe von Blättern und Blumen. Zu diesem summierten Potential tragen mindestens zwei Typen von Sensillen auf jedem Segment der Fühler bei, wobei allerdings die 6–8 Endglieder nicht proportional reagieren. Die Aktivitätsprofile einzelner Geruchszellen entsprechen einem typischen Spektrum eines generalist. 0–3 Zellen pro Sensille reagieren auf die Reizung mit erhöhter oder erniedrigter Impulsfrequenz. Die Markierung der Eier, welche andere Weibchen von der Eiablage abhält, ist in den Ovarien nicht vorhanden, wird jedoch in den akzessorischen Drüsen erzeugt. Sie ist wasserlöslich, hat eine geringe Flüchtigkeit und verursacht eine erhöhte Impulsfrequenz bei Kontaktrezeptoren der Tarsen.

     

The Steric Gate of DNA Polymerase ι Regulates Ribonucleotide Incorporation and Deoxyribonucleotide Fidelity

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A “steric gate” pol ι mutant is considerably more active in the presence of Mn²⁺ compared with Mg²⁺ and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be “at risk” for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.     

Early Adolescent Cognitive Gains Are Marked by Increased Sleep EEG Coherence

Although the increases in cognitive capacities of adolescent humans are concurrent with significant cortical restructuring, functional associations between these phenomena are unclear. We examined the association between cortical development, as measured by the sleep EEG, and cognitive performance in a sample of 9/10 year olds followed up 1 to 3 years later. Our cognitive measures included a response inhibition task (Stroop), an executive control task (Trail Making), and a verbal fluency task (FAS). We correlated sleep EEG measures of power and intra-hemispheric coherence at the initial assessment with performance at that assessment. In addition we correlated the rate of change across assessments in sleep EEG measures with the rate of change in performance. We found no correlation between sleep EEG power and performance on cognitive tasks for the initial assessment. In contrast, we found a significant correlation of the rate of change in intra-hemispheric coherence for the sigma band (11 to 16 Hz) with rate of change in performance on the Stroop (r = 0.61; p<0.02) and Trail Making (r = −0.51; p<0.02) but no association for the FAS. Thus, plastic changes in connectivity (i.e., sleep EEG coherence) were associated with improvement in complex cognitive function.     

Limnologic consequences of the decline in hemlock 4800 years ago in three Southern Ontario lakes

Four thousand eight hundred years ago hemlock (Tsuga canadensis) populations were decimated throughout eastern North America. We have studied the effects of this loss from the terrestrial community on three Southern Ontario lakes: Little Round Lake, Sunfish Lake, and McKay Lake. This study includes the use of cladocerans, diatoms, chrysophytes, and bacterial pigments to assess the limnologic changes that occurred in these lakes. Each lake experienced a change in trophic status that coincided with the loss of hemlock from its catchment, but the change in the aquatic biota was different in each lake. The lakes' size may have been the most influential factor governing the response to this terrestrial disturbance.     

Molecular genetic advances in tuberous sclerosis

Over the past decade, there has been considerable progress in understanding the molecular genetics of tuberous sclerosis, a disorder characterised by hamartomatous growths in numerous organs. We review this progress, from cloning and characterising TSC1 and TSC2, the genes responsible for the disorder, through to gaining insights into the functions of their protein products hamartin and tuberin, and the identification and engineering of animal models. We also present the first comprehensive compilation and analysis of all reported TSC1 and TSC2 mutations, consider their diagnostic implications and review genotype/phenotype relationships.     

The role of circulating serotonin in the development of chronic obstructive pulmonary disease

Background

Cigarette smoking is a major risk factor in the development of age-related chronic obstructive pulmonary disease (COPD). The serotonin transporter (SERT) gene polymorphism has been reported to be associated with COPD, and the degree of cigarette smoking has been shown to be a significant mediator in this relationship. The interrelation between circulating serotonin (5-hydroxytyptamine, 5-HT), cigarette smoking and COPD is however largely unknown. The current study aimed at investigating the mediation effects of plasma 5-HT on cigarette smoking-induced COPD and the relation between plasma 5-HT levels and age.

Methods

The association between plasma 5-HT, age and COPD was analyzed in a total of 62 COPD patients (ever-smokers) and 117 control subjects (healthy non-smokers and ever-smokers). Plasma 5-HT levels were measured by enzyme-linked immuno assay (EIA).

Results

The elevated plasma 5-HT levels were significantly associated with increased odds for COPD (OR = 1.221, 95% CI = 1.123 to 1.319, p<0.0001). The effect remained significant after being adjusted for age and pack-years smoked (OR = 1.271, 95% CI = 1.134 to 1.408, p = 0.0003). Furthermore, plasma 5-HT was found to mediate the relation between pack-years smoked and COPD. A positive correlation (r = 0.303, p = 0.017) was found between plasma 5-HT levels and age in COPD, but not in the control subjects (r = −0.149, p = 0.108).

Conclusion

Our results suggest that cigarette smoke-induced COPD is partially mediated by the plasma levels of 5-HT, and that these become elevated with increased age in COPD. The elevated plasma 5-HT levels in COPD might contribute to the pathogenesis of this disease.