Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms.

The microbial community of a denitrifying sand filter in a municipal wastewater treatment plant was examined by conventional and molecular techniques to identify the bacteria actively involved in the removal of nitrate. In this system, denitrification is carried out as the last step of water treatment by biofilms growing on quartz grains with methanol as a supplemented carbon source. The biofilms are quite irregular, having a median thickness of 13 to 20 microns. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp. in the sand filter. 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization. Stringency requirements for the probes were adjusted by use of a formamide concentration gradient to achieve complete discrimination of even highly similar target sequences. Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven of the 56 isolates hybridized with the genus-specific probes. In situ hybridization identified dense aggregates of paracocci in detached biofilms. Probes complementary to the type strains of P. denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting the presence of a new Paracoccus species in the sand filter. Analysis using confocal laser scanning microscopy detected spherical aggregates of morphologically identical cells exhibiting a uniform fluorescence. Cell quantification was performed after thorough disruption of the biofilms and filtration onto polycarbonate filters. An average of 3.5% of total cell counts corresponded to a Paracoccus sp., whereas in a parallel sand filter with no supplemented methanol, and no measurable denitrification, only very few paracocci (0.07% of cells stained with 4',6-diamidino-2-phenylindole) could be detected. Hyphomicrobium spp. constituted approximately 2% of all cells in the denitrifying unit and could not be detected in the regular sand filter. This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process. Possible selective advantages favoring the paracocci in this habitat are discussed.     

Octopamine mediates rapid stimulation of protein kinase A in the antennal lobe of honeybees

In the honeybee octopamine mediates mechanisms of arousal that interfere with the appetitive proboscis extension response to food-indicating chemosensory stimuli. This study demonstrates that injections of octopamine or cyclic adenosine monophosphate (cAMP) into the primary chemosensory neuropil of the honeybee, the antennal lobe, evokes a rapid and transient activation of cAMP-dependent protein kinase (PKA). Other monoamines detectable in the antennal lobe, dopamine and serotonin, do not affect the level of PKA activity. Stimulation of the bees' antenna with the appetitive stimulus water or sucrose solution in vivo also causes a short-term activation of PKA in the antennal lobe. The increased PKA activity can be detected immediately (0.5 s) after stimulation but reverts to the basal level within 3 s. This effect can be abolished by monoamine depletion with reserpine. Since octopamine is the only monoamine that stimulates PKA, it appears to mediate the PKA activation after sucrose stimulus and may contribute to the processing of this chemosensory input. © 1995 John Wiley & Sons, Inc.     

Synthesis of (+)-(R)- and (−)-(S)-5-hydroxy-2-methyl-2-dipropylaminotetralin: Effects on rat hippocampal output of 5-HT, 5-HIAA,and DOPAC as determined by in vivo microdialysis

Racemic 5-methoxy-2-methyl-2-dipropylaminotetralin ( 3 ) has been prepared by a short synthetic route, in which the N,N-dipropyliminium perchlorate of 5-methoxy-2-tetralone ( 4 ) is a key intermediate. Racemic 3 was resolved by crystallization of the corresponding diastereomeric di-p-toluoyltartrates. The enantiomeric excess (%ee) of the phenolic derivatives of (+)-(R)- and (?)-(S)-3 [(+)-(R)- and (?)-(S)-2] was determined by ¹HNMR spectroscopic analysis of the corresponding diastereomeric (?)-(R)-1,1′-binaphthyl-2,2′-diylphosphoric acid salts utilizing ¹³C satellites. X-ray crystallography established the absolute configuration of (?)-(S)-2 · HCl. The enantiomers of 2 were tested for hippocampal output of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and dihydroxyphenylacetic acid in rats by use of in vivo microdialysis. The (?)-(S)-enantiomer appeared to affect 5-HT-turnover, whereas (+)-(R)- 2 was inactive. Results obtained provide support for the previously reported hypothesis that the inactivity of (?)-(S)- 2 at central DA receptors is caused by the steric bulk of the C(2)-methyl group. This makes it possible to define a “DA D2 receptor essential volume.” © 1993 Wiley-Liss, Inc.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Analysis of penicillin-binding sites with a micro method — The binding site of PBP 5 from Escherichia coli

Abstract A micro method for the isolation and characterization of the penicillin-binding sites in penicillin-binding proteins (PBPs) was developed. Only 10 nmol of a pure PBP are required for the whole procedure which is based on high-pressure liquid chromatography (HPLC). We showed that serine 44 in PBP 5 from Escherichia coli binds penicillin covalently.     

Chronopharmacokinetics and Cardiovascular Effects of Nifedipine

Orcadian phase dependency in pharmacokinetics and hemodynamic effects on blood pressure and heart rate of different galenic formulations of nifedipine (immediate-release, sustained-release, and i.v. solution) were studied in healthy subjects or in hypertensive patients. Pharmacokinetics of immediate-release but not sustained-release and i.v. nifedipine were dependent on time of day: immediate-release nifedipine had higher C_max (peak concentration) and shorter t_max (time-to-peak concentration) after morning than evening application, and bioavailibility in the evening was reduced by about 40%. Orcadian rhythm in estimated hepatic blood flow as determined by indocyanine green kinetics may contribute to these chronokinetics. A circadian time dependency was also found in nifedipine-induced effects on blood pressure and heart rate as monitored by 24-h ambulatory blood pressure measurements. In conclusion, the dose response relationship of oral nifedipine is influenced by the circadian organization of the cardiovascular system as well as by the galenic drug formulation.     

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.     

Effect of NO Synthase Inhibition on Cardiovascular Circadian Rhythms in Wild‐Type and eNOS‐Knock‐Out Mice

Cardiovascular functions (blood pressure [BP], heart rate [HR]) were radiotelemetrically studied in endothelial nitric oxide synthase (NOS) knock‐out mice (eNOS‐/‐) and their wild type C57BL/6 (WT) controls. Studies were performed with and without inhibition of the NOS with the non‐specific inhibitor N^ω‐Nitro‐L‐Arginin‐Methylester (L‐NAME). Six eNOS‐/‐and five WT mice, kept under a light:dark schedule of 12:12 h (lights on 07:00 h), were treated with L‐NAME in tap water containing different concentrations (94, 282, and 940 mg/kg) each for three days. Under control conditions, the eNOS‐/‐mice are mildly hypertensive in comparison to WT. L‐NAME increased systolic [SBP] and diastolic [DBP] blood pressures in WT mice to the levels of eNOS‐/‐mice after two days of L‐NAME application with no dose‐dependency, whereas L‐NAME had no effects on SBP and DBP in eNOS‐/‐mice. In neither mouse strain were the circadian rhythms in BP and HR affected by drug treatment. The similarity of the 24 h BP profiles in eNOS‐/‐and L‐NAME‐treated WT mice support the notion that only the enothelial NOS and not other NOS isoenzymes are of importance for hypertension in the knock‐out mouse strain.     

Familial Sinistrals Avoid Exact Numbers

We report data from an internet questionnaire of sixty number trivia. Participants were asked for the number of cups in their house, the number of cities they know and 58 other quantities. We compare the answers of familial sinistrals – individuals who are left-handed themselves or have a left-handed close blood-relative – with those of pure familial dextrals – right-handed individuals who reported only having right-handed close blood-relatives. We show that familial sinistrals use rounder numbers than pure familial dextrals in the survey responses. Round numbers in the decimal system are those that are multiples of powers of 10 or of half or a quarter of a power of 10. Roundness is a gradient concept, e.g. 100 is rounder than 50 or 200. We show that very round number like 100 and 1000 are used with 25% greater likelihood by familial sinistrals than by pure familial dextrals, while pure familial dextrals are more likely to use less round numbers such as 25, 60, and 200. We then use Sigurd’s (1988, Language in Society) index of the roundness of a number and report that familial sinistrals’ responses are significantly rounder on average than those of pure familial dextrals. To explain the difference, we propose that the cognitive effort of using exact numbers is greater for the familial sinistral group because their language and number systems tend to be more distributed over both hemispheres of the brain. Our data support the view that exact and approximate quantities are processed by two separate cognitive systems. Specifically, our behavioral data corroborates the view that the evolutionarily older, approximate number system is present in both hemispheres of the brain, while the exact number system tends to be localized in only one hemisphere.