Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.     

Molecular phylogeny of the crab genus Brachynotus (Brachyura: Varunidae) based on the 16S rRNA gene

The crab genus Brachynotus de Haan, 1833 is restricted to the intertidal and shallow subtidal of the Mediterranean and northeastern Atlantic. It is presently recognized to consist of four species, of which three (B. foresti, B. gemmellari and B. sexdentatus) are endemic to the Mediterranean. The fourth species, B. atlanticus, is found along the Atlantic coasts of northern Africa and southern Europe, but also extends into the western Mediterranean. This high level of endemism suggests that speciation within Brachynotus is strongly correlated with the geography and geology of the Mediterranean Sea. A molecular phylogeny based on the mitochondrial large subunit (16S) rRNA gene indicates that the four species of Brachynotus form a monophyletic group within Atlantic Varunidae. The DNA sequence data also show that the genus Brachynotus can be subdivided into two species groups, one comprising B. atlanticus and B. foresti, and the other one B. gemmellari and B. sexdentatus. While B. atlanticus and B. foresti are clearly genetically distinct, B. gemmellari and B. sexdentatus are identical in the studied region of the 16S rRNA gene, suggesting a recent separation or continuing gene flow.     

Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-(13)C(2)]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.     

Epidemiology of Rhodococcus equi Strains on Thoroughbred Horse Farms

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.     

Effects of Passage Through Tamarin Guts on the Germination Potential of Dispersed Seeds

Passage through tamarin guts may have an effect on seed germination potential. To examine these effects, and the variation between 2 sympatric tamarin species, we studied Saguinus mystax and S. fuscicollis in northeastern Peruvian Amazonia. For most of 39 plant species, neither germination success nor latency was modified by gut passage. Neutral effects on seed germination potential suggest that tamarins may fulfill criteria for effective seed dispersal.     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Weight Loss Increases Soluble Leptin Receptor Levels and the Soluble Receptor Bound Fraction of Leptin

Objective: Soluble leptin receptor (sOB‐R) represents the main binding site for leptin in human blood. The aim of this study was to investigate the relationship between leptin and soluble leptin receptor and the bound/free ratio after pronounced weight reduction. Research Methods and Procedures: A total of 18 morbidly obese women participated in this prospective study. Subjects were examined for fat mass, leptin, and sOB‐R concentrations before and 1 year after Swedish adjustable gastric banding. Results: Anthropomorphic measures displayed a significant reduction of body mass index [(42.9 ± 5.6 to 32.9 ± 6.0 kg/m² (mean ± SD)]. Fat mass decreased from 56.3 ± 9.0 to 33.9 ± 12.5 kg. Plasma leptin concentration decreased from 44.6 ± 18.0 to 20.0 ± 13.1 ng/mL (p < 0.001), whereas the sOB‐R levels increased from 11.1 ± 3.6 to 16.6 ± 6.0 U/mL after weight‐reducing surgery. Thus, the sOB‐R bound fraction of leptin increased from 7% to 33%. Discussion: This work demonstrates a relationship between weight loss, leptin, and sOB‐R concentrations in vivo. During weight loss, leptin levels decreased, whereas sOB‐R levels and the receptor bound fraction of leptin increased. Thus, sOB‐R may negatively regulate free leptin.     

A comparison of four different fine root production estimates with ecosystem carbon balance data in a Fagus–Quercus mixed forest

The controversy on how to measure fine root production of forests (P) most accurately continues. We applied four different approaches to determine annual rates of P in an old-growth temperate Fagus sylvatica–Quercus petraea stand: sequential soil coring with minimum–maximum calculation, sequential coring with compartmental flow calculation, the ingrowth core method, and a recently developed root chamber method for measuring the growth of individual fine roots in situ. The results of the four destructive approaches differed by an order of magnitude and, thus, are likely to introduce large errors in estimating P. The highest annual rates of P were obtained from the sequential coring approach with compartmental flow calculation, intermediate rates by sequential coring with minimum–maximum calculation, and low ones by both the root growth chamber and ingrowth core approaches. A carbon budget for the stand was set up based on a model of annual net carbon gain by the canopy and measurements on carbon sink strength (annual leaf, branch and stem growth). The budget implied that a maximum of 27% of the net carbon gain was available for allocation to fine root growth. When compared to the carbon budget data, the sequential coring/compartmental flow approach overestimated annual fine root production substantially; whereas the ingrowth core and root growth chamber approaches grossly underestimated P rates. With an overestimation of about 25% the sequential coring/minimum–maximum approach demonstrated the best agreement with the carbon budget data. It is concluded that the most reliable estimate of P in this temperate forest will be obtained by applying the sequential coring/minimum–maximum approach, conducted with a large number of replicate samples taken on a few dates per season, in conjunction with direct root growth observation by minirhizotrons.     

Project NEMESIS: perception of a 50 Hz electric and magnetic field at low intensities (laboratory experiment).

The Electrical Hypersensitivity Syndrome (EHS) is a condition where people suffer from various nonspecific health symptoms attributed to an assumed adverse effect of electric and magnetic fields (EMF). Many EHS patients report the ability to consciously perceive EMF at very low intensities. The existence of a direct EMF perception could be the key to explain at least partially the aetiology of EHS through stress mechanisms and allow the comparison with well known environmental stressors such as noise or odor. The double blind laboratory experiment tested the hypothesis that there are subjects with the ability to perceive 50 Hz EMF at 100 V/m and 6 microT (EMF sensitive) and to investigate the prevalence of EMF sensitivity in a group consisting of subjects with or without self-reported EHS. A total of 63 volunteers, 49 with EHS and 14 controls, took part in the EMF perception experiment, where 10 sham and 10 exposed 2 min blocks had to be judged in randomized sequence (field on/field off). Seven out of 63 subjects reached a statistically significant result which points to the existence of a small EMF sensitive subgroup within the study group. There was no relevant difference between the subjects with self reported EHS and those without in terms of the success rate in the field perception experiment, as well as the number and types of symptoms encountered during the test. The results of the EMF perception experiment suggest that EHS is not a prerequisite for the ability to consciously perceive weak EMF and vice versa.