Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

Ultrastructural changes induced by alpha-sarcin in a human pulmonary tumor grown in naked mice.

alpha-Sarcin is a cytotoxic polypeptide produced by Aspergillus giganteus. It suppresses protein synthesis in yeast and wheat germ extracts and has a purine-specific RNase activity. The substance has been tested for its antitumor properties in a series of induced tumor systems in mice such as sarcoma and carcinoma among others. Although some of the in vitro effects of alpha-Sarcin on certain cellular components have been elucidated, the biological effects leading to cellular damage are still obscure. In this work we analysed the morphological changes in tumor cells derived from human pulmonary adenocarcinoma heterotransplanted and grown in naked mice, induced shortly (24 hours) after a single intratumoral injection of alpha-Sarcin (0.4 mg/tumor). The results obtained were: 1) swelling of mitochondria; 2) cell necrosis with partial removal of necrotic cells by phagocytosis; 3) thickening of interlobular connective tissue; 4) hyperplasia of goblet-cell-like clear cells. The mode of action concerning these cellular changes is presently uncertain. In view of the severity of these structural alterations it seems conceivable that alpha-Sarcin may enter the cell undergoing interactions with different intracellular structures. This would require a selective membrane permeabilization, perhaps induced upon formation of complexes with negatively-charged membrane phospholipids.     

The atokous-epitokous border is determined before the onset of heteronereid development in Platynereis dumerilii (Annelida,Polychaeta)

Summary In most nereids sexual maturation is accompanied by a dramatic reorganization of the body that enables swarming of the formerly benthic worms. However, a border exists between unchanged anterior (atokous) and metamorphosed posterior (epitokous) segments. The site of this atokous-epitokous border (a/e border) is different in sexually mature males and females of Platynereis dumerilii. There is no correlation between the total number of setigerous segments of a specimen and the location of the a/e border. The location of the a/e border and sexual development are affected neither by cutting off caudal segments of juveniles (including the prospective a/e border) nor by transecting the ventral nerve cord. When parapodia are transplanted from prospective epitokous regions to prospective atokous regions and vice versa, they maintain their original character during metamorphosis. The results presented here suggest that prospective atokous as well as epitokous characters are determined at or only very shortly after formation of the respective segments. Thus the a/e border is established well in advance of the onset of epitokous metamorphosis.     

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca²⁺/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca²⁺/calmodulin, one at submicromolar Ca²⁺ concentrations and one in the micromolar Ca²⁺ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca²⁺/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca²⁺ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca²⁺ regulation in anoctamin Cl⁻ channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.     

Effects of alcohol mixed with energy drink and alcohol alone on subjective intoxication

Recent studies suggest that the combination of caffeine-containing drinks together with alcohol might reduce the subjective feelings of alcohol intoxication—the so-called “masking effect”. In this study, we aimed to review the effects of alcohol in combination with caffeine or energy drink with special focus on the “masking effect”. Fifty-two healthy male volunteers were analysed concerning breath alcohol concentration and subjective sensations of intoxication using a 18 item Visual Analogue Scale in a randomised, double-blinded, controlled, four treatments cross-over trial after consumption of (A) placebo, (B) alcohol (vodka 37.5 % at a dose of 46.5 g ethanol), (C) alcohol in combination with caffeine at a dose of 80 mg (equivalent to one 250 ml can of energy drink) and (D) alcohol in combination with energy drink at a dose of 250 ml (one can). Primary variables were headache, weakness, salivation and motor coordination. Out of four primary variables, weakness and motor coordination showed a statistically significant difference between alcohol and non-alcohol group, out of 14 secondary variables, five more variables (dizziness, alterations in sight, alterations in walking, agitation and alterations in speech) also showed significant differences due mainly to contrasts with the non-alcohol group. In none of these end points, could a statistically significant effect be found for the additional ingestion of energy drink or caffeine on the subjective feelings of alcohol intoxication. This within-subjects study does not confirm the presence of a “masking effect” when combining caffeine or energy drink with alcohol.     

The Bacterial SMC Complex Displays Two Distinct Modes of Interaction with the Chromosome

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Preeclampsia and Blood Pressure Trajectory during Pregnancy in Relation to Vitamin D Status

Every tenth pregnancy is affected by hypertension, one of the most common complications and leading causes of maternal death worldwide. Hypertensive disorders in pregnancy include pregnancy-induced hypertension and preeclampsia. The pathophysiology of the development of hypertension in pregnancy is unknown, but studies suggest an association with vitamin D status, measured as 25-hydroxyvitamin D (25(OH)D). The aim of this study was to investigate the association between gestational 25(OH)D concentration and preeclampsia, pregnancy-induced hypertension and blood pressure trajectory. This cohort study included 2000 women. Blood was collected at the first (T1) and third (T3) trimester (mean gestational weeks 10.8 and 33.4). Blood pressure at gestational weeks 10, 25, 32 and 37 as well as symptoms of preeclampsia and pregnancy-induced hypertension were retrieved from medical records. Serum 25(OH)D concentrations (LC-MS/MS) in T1 was not significantly associated with preeclampsia. However, both 25(OH)D in T3 and change in 25(OH)D from T1 to T3 were significantly and negatively associated with preeclampsia. Women with a change in 25(OH)D concentration of ≥30 nmol/L had an odds ratio of 0.22 (p = 0.002) for preeclampsia. T1 25(OH)D was positively related to T1 systolic (β = 0.03, p = 0.022) and T1 diastolic blood pressure (β = 0.02, p = 0.016), and to systolic (β = 0.02, p = 0.02) blood pressure trajectory during pregnancy, in adjusted analyses. There was no association between 25(OH)D and pregnancy-induced hypertension in adjusted analysis. In conclusion, an increase in 25(OH)D concentration during pregnancy of at least 30 nmol/L, regardless of vitamin D status in T1, was associated with a lower odds ratio for preeclampsia. Vitamin D status was significantly and positively associated with T1 blood pressure and gestational systolic blood pressure trajectory but not with pregnancy-induced hypertension.