Unexpected Accumulation of ncm\u3csup\u3e5\u3c/sup\u3eU and ncm\u3csup\u3e5\u3c/sup\u3es\u3csup\u3e2\u3c/sup\u3eU in a \u3cem\u3etrm9\u3c/em\u3e Mutant Suggests an Additional Step in the Synthesis of mcm\u3csup\u3e5\u3c/sup\u3eU and mcm\u3csup\u3e5\u3c/sup\u3es\u3csup\u3e2\u3c/sup\u3eU

Background Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-methoxycarbonylmethyl-uridine (mcm5U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The formation of mcm5 and ncm5 side chains involves a complex pathway, where the last step in formation of mcm5 is a methyl esterification of cm5 dependent on the Trm9 and Trm112 proteins. Methodology and Principal Findings Both Trm9 and Trm112 are required for the last step in formation of mcm5 side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm5U or mcm5s2U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm5U and mcm5s2U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm5U and ncm5s2U, not the expected cm5U and cm5s2U. Conclusions Trm9p and Trm112p function together at the final step in formation of mcm5U in tRNA by using the intermediate cm5U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm5U and ncm5s2U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm5 side chain. We propose two alternative explanations for this observation. One is that the intermediate cm5U is generated from ncm5U by a yet unknown mechanism and the other is that cm5U is formed before ncm5U and mcm5U

Kluyveromyces lactis γ-toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA

Kluyveromyces lactis γ-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae tRNAmcm5s2UUCGlu3, tRNAmcm5s2UUULys and tRNAmcm5s2UUGGln between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) residue at position 34 (wobble position) of which the mcm5 group is required for efficient cleavage. However, the different cleavage efficiencies of mcm5s2U34-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by γ-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate tRNAmcm5s2UUCGlu3 is cleaved at the same position as the natural substrate. In ASLUUCGlu3, the nucleotides U34U35C36A37C38 are required for optimal γ-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast tRNAUUCGlu reinforced the strong stimulatory effects of the mcm5 group, revealed a weak positive effect of the s2 group and a negative effect of the bacterial 5-methylaminomethyl (mnm5) group. The data underscore the high specificity of this yeast tRNA toxin

Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue

BACKGROUND: Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. METHODS: Uterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. RESULTS: In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to non-pregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy. CONCLUSION: The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility

mRNA expression and localization of bNOS, eNOS and iNOS in human cervix at preterm and term labour

BACKGROUND: Preterm birth is the primary cause of the neonatal mortality and morbidity. There will be no preterm birth without a cervical softening. Nitric oxide (NO) is shown to be a mediator of term cervical ripening. The aim of this study was to investigate mRNA expression of the three isomers of NO synthases (NOS) and to identify them by immunohistochemistry in the human cervix at preterm birth compared to term. METHODS: The three isomers of NOS- inducible (iNOS), endothelial (eNOS) and neuronal (bNOS) – were investigated in the human cervix. The expression of mRNA was determined using Real-Time Multiplex RT-PCR. The localisation of synthases in the cervical tissue was analysed using immunohistochemistry. Cervical biopsies were obtained from 4 groups of women without clinical signs of infection: preterm (PTL), term labour (TL), preterm not in labour (PTnotL) and term not in labour (TnotL) patients. One-Way ANOVA, Kruskal-Wallis, Student t-test or Mann-Whitney test were applied as appropriate to determine statistically significant differences among the groups. RESULTS: Patients in preterm labour had significantly (p < 0.01) higher mRNA levels of all the three NOS isomers compared to those in term labour. Women not in labour, irrespective of gestational age, thus with unripe cervices, had significantly lower eNOS mRNA levels compared to those in labour (p < 0.01). Immunoreactivity for all three NO synthases was observed in each examined sample in all groups. The bNOS staining was the most prominent. CONCLUSION: The mRNA levels were higher in the preterm labour group compared to the women at term labour. The significant increase of the eNOS mRNA expression, from the unripe to the favourable cervical state during labour, may indicate a role of eNOS and supports the role of NO in the cervical ripening process. All the three synthases were identified by immunohistochemistry in all the groups of study

Linkage between fitness of yeast cells and adenylate kinase catalysis

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies

Temperature and Resource Availability May Interactively Affect Over-Wintering Success of Juvenile Fish in a Changing Climate

The predicted global warming may affect freshwater systems at several organizational levels, from organism to ecosystem. Specifically, in temperate regions, the projected increase of winter temperatures may have important effects on the over-winter biology of a range of organisms and especially for fish and other ectothermic animals. However, temperature effects on organisms may be directed strongly by resource availability. Here, we investigated whether over-winter loss of biomass and lipid content of juvenile roach (Rutilus rutilus) was affected by the physiologically relatively small (2-5°C) changes of winter temperatures predicted by the Intergovernmental Panel on Climate Change (IPCC), under both natural and experimental conditions. This was investigated in combination with the effects of food availability. Finally, we explored the potential for a correlation between lake temperature and resource levels for planktivorous fish, i.e., zooplankton biomass, during five consecutive winters in a south Swedish lake. We show that small increases in temperature (+2°C) affected fish biomass loss in both presence and absence of food, but negatively and positively respectively. Temperature alone explained only a minor part of the variation when food availability was not taken into account. In contrast to other studies, lipid analyses of experimental fish suggest that critical somatic condition rather than critical lipid content determined starvation induced mortality. Our results illustrate the importance of considering not only changes in temperature when predicting organism response to climate change but also food-web interactions, such as resource availability and predation. However, as exemplified by our finding that zooplankton over-winter biomass in the lake was not related to over-winter temperature, this may not be a straightforward task

Defects in tRNA Modification Associated with Neurological and Developmental Dysfunctions in Caenorhabditis elegans Elongator Mutants

Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5′methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development

Evaluation of endodontic treatment of teeth with apical periodontitis

Apical periodontitis, an acute or chronic inflamination around the apex of the tooth, is caused by bacteria in the root canal. In Sweden the dentists devote around 10X of their total time to treating this disease. The treatment usually requires 3 to 5 sessions. The treatment may fail in up to 25X of the cases. In the present study various treatment regimens were evaluated. One hundred and forty singlerooted teeth with apical periodontitis were treated. The importance of mechanical instrumentation, irrigating solutions and antibacterial dressings in eliminating bacteria from the infected root canals was studied using bacteriological techniques. The healing of the apical periodontitis after treatment was followed for 2 to 5 years on recall radiographs. Bacteria were found in all 140 root canals at the beginning of the treatment. Most of these bacteria were anaerobes and they represented a restricted group of bacteria compared to the bacteria present at other sites in the oral cavity. Mechanical instrumentation with files and reamers in combination with saline irrigation reduced the number of bacterial cells in the root canal 100- to 1000-fold during one treatment session. Bacteria could be eliminated from about half the number of root canals if this treatment was performed at 4 sessions. Mechanical instrumentation and irrigation with 0.5X or 5X sodium hypochlorite solutions or with the 5X solution in combination with 15X EDTA solution wa3 more efficient and the bacteria were eliminated from about half the treated canals after one treatment session. The bacteria which persisted in the root canal after this treatment usually increased in number during the interval up to the next session and reached levels which were often as high as in the initial sample at the previous session. All bacteria persistent in the root canals after the previous treatment regimens were with 2 exceptions eliminated by dressing the root canals for 1 to 2 months with calcium hydroxide paste. Thirty-four out of 35 root canals treated at the first session with mechanical instrumentation, irrigation with sodium hypochlorite solution and dressed with calcium hydroxide paste were free of bacteria at the second session. Calcium hydroxide paste was superior to camphorated phenol and camphorated paramonochlorophenol as dressing. Healing of 79 out of the 140 treated teeth was followed for 2 to 5 years. The majority of the lesions healed completely or decreased in size in such a way that they could be expected to heal. There was no or only an insignificant decrease in the size of the lesions in 5 cases. In 2 of these cases bacteria were demonstrated in the periapical tissues and in a third case dentin chips. Periapical lesions may thus fail to heal in a few cases due to an establishment of bacteria outside the root canal, and in that site the bacteria are inaccessible to conventional endodontic treatment. The present study showed that treatment of the majority of infected non-vital teeth can be completed in only 2 sessions, if mechanical instrumentation, sodium hypochlorite irrigation and calcium hydroxide dressing are combined.Diss. (sammanfattning) Umeå : Umeå universitet, 1986, härtill 5 uppsatser.digitalisering@um