The plasma membrane of Streptococcus cremoris: isolation and partial characterization

Plasma membrane was isolated from Streptococcus cremoris using mutanolysin from a streptomycete as the cell wall-degrading enzyme and phenylmethylsulfonyl fluoride as protease inhibitor. The specific activity of membrane-bound enzyme, adenosine triphosphatase (ATPase), was 4 μmol/mg protein per min, which was 5–10 times higher than the activity found in other fractions obtained during the isolation procedure. The number of polypeptides in the plasma membrane was approximately 50 with molecular weights 13 500–100 000, minor changes in the polypeptide pattern were observed when the plasma membrane was isolated without a protease inhibitor. The chemical composition of the membrane preparation was 49.7% protein, 21.9% lipid, 5.1% aminosugars, 17.3% RNA and 0.03% DNA. Electron microscopic examination confirmed the membrane to be practically devoid of cell wall components. Our results indicate that the membrane integrity is well retained and therefore the membrane preparation is suitable for detailed studies on vectorial metabolism and its enzymes, e.g. ATPase.     

Immunoreactivity for substance P in the gasserian ganglion, ophthalmic nerve and anterior segment of the rabbit eye

Summary The distribution of substance P (SP) immunofluorescence was investigated in the Gasserian ganglion, ophthalmic nerve and in the anterior segment of the rabbit eye. About one third of the nerve cell bodies in the Gasserian ganglion exhibited SP immunofluorescence, which was also observed in some nerve fibres of the ophthalmic nerve. In the cornea, some SP-positive iris contained numerous nerve fibres with SP immunofluorescence. In the sphincter area such fibres were circular, while the orientation of the SP fibres was radial in the dilator muscle. Both in the iris and in the ciliary body, the largest vessels were surrounded by nerves exhibiting SP immunofluorescence. A few nerve fibres also appeared in the stroma of the ciliary processes.     

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.     

Employing a Monte Carlo Algorithm in Newton-Type Methods for Restricted Maximum Likelihood Estimation of Genetic Parameters

Estimation of variance components by Monte Carlo (MC) expectation maximization (EM) restricted maximum likelihood (REML) is computationally efficient for large data sets and complex linear mixed effects models. However, efficiency may be lost due to the need for a large number of iterations of the EM algorithm. To decrease the computing time we explored the use of faster converging Newton-type algorithms within MC REML implementations. The implemented algorithms were: MC Newton-Raphson (NR), where the information matrix was generated via sampling; MC average information(AI), where the information was computed as an average of observed and expected information; and MC Broyden''s method, where the zero of the gradient was searched using a quasi-Newton-type algorithm. Performance of these algorithms was evaluated using simulated data. The final estimates were in good agreement with corresponding analytical ones. MC NR REML and MC AI REML enhanced convergence compared to MC EM REML and gave standard errors for the estimates as a by-product. MC NR REML required a larger number of MC samples, while each MC AI REML iteration demanded extra solving of mixed model equations by the number of parameters to be estimated. MC Broyden''s method required the largest number of MC samples with our small data and did not give standard errors for the parameters directly. We studied the performance of three different convergence criteria for the MC AI REML algorithm. Our results indicate the importance of defining a suitable convergence criterion and critical value in order to obtain an efficient Newton-type method utilizing a MC algorithm. Overall, use of a MC algorithm with Newton-type methods proved feasible and the results encourage testing of these methods with different kinds of large-scale problem settings.     

COASTAL DIATOM–ENVIRONMENT RELATIONSHIPS FROM THE GULF OF FINLAND,BALTIC SEA1

Eutrophication of the Baltic Sea has become a serious concern in recent decades. To provide a potential means for quality assessments of coastal waters in this area, we collected a data set of 49 embayments in the Gulf of Finland, and explored the relationship between surface sediment diatom assemblages and 15 environmental variables, with special emphasis on nutrients. Total dissolved nitrogen, total phosphorus, depth, and salinity all accounted for significant and independent fractions of variation in the diatom data and explained 34% of the total variation. There were clear changes in diatom assemblage structures along the nutrient gradients. Although these changes were gradual, we could identify a number of taxa that were more abundant in a particular nutrient environment. These taxa could be used as potential indicators of the quality of coastal waters in the Baltic Sea. Diatom assemblages that were least affected by nutrient enrichment included a variety of benthic species and a relatively high species richness. Small planktonic taxa such as Cyclotella atomus Hustedt, Cyclotella meneghiniana Kützing and Thalassiosira pseudonana Hasle and Heimdal were good indicators of highly elevated nutrient concentrations (>600 lg·L^?1 total dissolved nitrogen and 60 lg·L^?1 total phosphorus) together with low species richness. The first appearance of these small planktonic taxa in regular monitoring could be used as an early warning sign for deteriorating water quality. Diatoms could be applied to water quality classification and monitoring purposes in the coastal waters of the Baltic Sea area using techniques such as weighted‐averaging regression and calibration.     

Identification of hepatotoxin-producing cyanobacteria by DNA-chip

We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena , Microcystis , Planktothrix , Nostoc and Nodularia . Genus-specific probe pairs were designed for the detection of the microcystin ( mcyE ) and nodularin synthetase genes ( ndaF ) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE / ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE / ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1–5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.     

Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90

The cluster of microcystin synthetase genes from Anabaena strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (mcyA-mcyB-mcyC) is transcribed in the opposite direction from the second operon (mcyG-mcyD-mcyJ-mcyE-mcyF-mcyI) and the third operon (mcyH). The genes mcyA, mcyB, and mcyC encode nonribosomal peptide synthetases (NRPS), while mcyD codes for a polyketide synthase (PKS), and mcyG and mcyE are mixed NRPS-PKS genes. The genes mcyJ, mcyF, and mcyI are similar to genes coding for a methyltransferase, an aspartate racemase, and a D-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of mcyB coding for the adenylation domain was found to be 96% identical with the corresponding part of mcyC, suggesting a recent duplication of this fragment and a replacement in mcyB. In Anabaena strain 90, the order of the domains encoded by the genes in the two sets (from mcyG to mcyI and from mcyA to mcyC) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in Anabaena strain 90 differs from the arrangement found in two other cyanobacterial species, Microcystis aeruginosa and Planktothrix agardhii. The average sequence match between the microcystin synthetase genes of Anabaena strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.     

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     

Enolases from Gram-positive bacterial pathogens and commensal lactobacilli share functional similarity in virulence-associated traits

Enolase occurs as a cytoplasmic and a surface-associated protein in bacteria. Enolases of the bacterial pathogens Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus, as well as of the commensal lactic acid bacteria, Lactobacillus crispatus and Lactobacillus johnsonii, were purified as His(6)-fusion proteins from recombinant Escherichia coli. The fusion proteins were compared for putative virulence-associated functions, i.e., binding of human plasminogen, enhancement of plasminogen activation by human plasminogen activators, as well as binding to immobilized laminin, fibronectin and collagens. The individual enolases showed varying efficiencies in these functions. In particular, highly and equally effective interactions with plasminogen and laminin were seen with lactobacillar and staphylococcal enolases.