Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

Accumulation of pyrophosphate correlates with the increased level of nucleoside triphosphates in Escherichia coli

We measured the concentration of nucleoside triphosphates and inorganic pyrophosphate in Escherichia coli in conditions where nucleotide synthesis or nucleic acid synthesis was inhibited. The inhibitors that brought about an accumulation of some of the four ribonucleoside triphosphates also increased the pyrophosphate level. In a pyrimidine auxotrophic strain uracil starvation led to simultaneous accumulation of ATP and pyrophosphate, and they both rapidly returned to normal level when starvation was relieved. These results indicate the possible involvement of pyrophosphate in the reactions leading to the accumulation of nucleoside triphosphates.     

Effect of denucleation and UV-irradiation on the subcellular morphology inMicrasterias

Summary The effects of the elimination of the nuclear control on the ultrastructure of the green algaMicrasterias torreyi. Bail, have been studied by using centrifugation for denucleation and lethal dose of UV-light. Centrifugated anucleate cells were fixed 7 and 26 hours after the treatment and the UV-treated cells 4 and 8 hours after the irradiation. Although both treatments eliminate the nuclear control and the treated cells resemble morphologically each other, yet there are differences in ultrastructure suggesting that they are also brought about by other factors than the presence of nucleus. Both the treatments cause accumulation of cell wall material in the tips of lobes. The cell wall shows unusual secondary thickening with electron dense spots embedded in the matrix. The denucleation retards the functional cycle of Golgi apparatus and the production of vesicles has stopped in the 26-hour-denucleated cells. It is possible that flat vesicle production is totally absent in denucleated cells.First the UV-treatment seems to activate the function of Golgi apparatus but later on the vesicle production almost stops. It seems to eliminate the production of large vesicles but not that of dark vesicles.Both the treatments cause deterioration of ER membranes and polysomes, and in consequence, probably inhibit protein synthesis.Unlike UV-irradiation, denucleation appears to destroy the microtubule system. Mitochondrial cristae have almost entirely vanished within 26 hours after denucleation. Effect of denucleation and UV-irradiation on the subcellular morphology inMicrasterias.     

Family-Based Cluster Randomized Controlled Trial Enhancing Physical Activity and Motor Competence in 4–7-Year-Old Children

Little is known of how to involve families in physical activity (PA) interventions for children. In this cluster randomized controlled trial, we recruited families with four- to seven-year-old children to participate in a year-long study where parents in the intervention group families (n = 46) received tailored counseling to increase children’s PA. Structured PA was not served. Control group families (n = 45) did not receive any counseling. PA in all children (n = 91; mean age 6.16 ± 1.13 years at the baseline) was measured by accelerometers at the baseline and after three, six, nine and 12 months. Motor competence (MC) (n = 89) was measured at the baseline and after six and 12 months by a KTK (KörperkoordinationsTest für Kinder) and throwing and catching a ball (TCB) protocols. The effect of parental counseling on study outcomes was analyzed by a linear mixed-effects model fit by REML and by a Mann-Whitney U test in the case of the TCB. As season was hypothesized to affect counseling effect, an interaction of season on the study outcomes was examined. The results show significant decrease of MVPA in the intervention group when compared to the control group (p < .05). The TCB showed a nearly significant improvement at six months in the intervention group compared to the controls (p = .051), but not at 12 months. The intervention group had a steadier development of the KTK when the interaction of season was taken into account. In conclusion, more knowledge of family constructs associating with the effectiveness of counseling is needed for understanding how to enhance PA in children by parents. However, a hypothesis may be put forward that family-based counseling during an inactive season rather than an active season may provide a more lasting effect on the development of KTK in children.

Trial Registration

Controlled-Trials.com ISRCTN28668090     

Identification of keto acids in arctic bramble,Rubus arcticus L. as methyl esters of their 2,4-dinitrophenylhydrazones

The major keto acids in arctic bramble,Rubus arcticus L. were investigated. The acids were isolated with anionic and cationic ion-exchange resins, converted to 2,4-dinitrophenylhydrazones, and purified with an Al₂O₃ column. The derivatives were separated on a silica gel G thin-layer plate and esterified with methanol-HCl and the methyl esters of the keto acid 2,4-dinitrophenylhydrazones formed were analyzed on an OV-1-glass capillary gas-liquid chromatography column and with mass spectrometry. 2-Oxoglutaric, pyruvic, oxaloacetic, and glyoxylic acids were identified. The mass spectra of the derivatives are presented.     

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ～650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.