Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Groundwater and Aquifer Microorganisms

To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.     

Biosynthesis of isotopically labeled gramicidins and tyrocidins by Bacillus brevis

The three-dimensional structure of bilayer-associated gramicidin A is available from a structural data base. This and related peptides are, therefore, ideal model compounds to use during the implementation and development of new NMR techniques for the structural investigations of membrane proteins. As these methods rely on the isotopic labelling of single, selected or all sites, we have, investigated and optimised biochemical protocols using different strains of the Gram-positive bacterium Bacillus brevis. With newly developed schemes for isotopic labelling large amounts of gramicidin and tyrocidin enriched with stable isotopes such as ¹⁵N or ¹⁵N/¹³C have been obtained at low cost. A variety of analytical and spectroscopic techniques, including HPLC, mass spectrometry and NMR spectroscopy are used to characterise the resulting products.     

Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth<Emphasis Type="Italic"> Antheraea polyphemus</Emphasis>

SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further suggested SNMP-1 was expressed in only one of two to three neurons in male-specific pheromone-sensitive trichoid sensilla. To better understand the expression and localization of SNMP-1, an immunocytochemical study was performed using electron microscopy to visualize the distribution of SNMP-1 among the neurons of several classes of olfactory sensilla of both male and female antennae of the silkmoth Antheraea polyphemus. SNMP-1 antigenicity was primarily restricted to the receptive dendritic membranes of ORNs of all sensilla types examined and was observed in cytosolic granules, but not plasma membranes, of the cell soma. Mean labeling densities ranged from 1 to 16 gold particles per micrometer of dendrite circumference; dendrites of trichoid and intermediate sensilla showed significantly higher labeling densities than those of basiconic sensilla. Larger dendrites of trichoid sensilla showed significantly higher mean labeling densities (13-16/micron) than smaller diameter dendrites (3-7/micron). Immunofluorescence studies using baculovirus expressed SNMP-1 and multiphoton photon laser scanning microscopy (MPLSM) indicated that rSNMP-1, which was post-translationally processed to the in vivo molecular weight, was inserted into the plasma membrane in a topography presenting extracellular epitopes. These studies suggest SNMP-1 is a common feature of the ORNs, is asymmetrically expressed among functionally distinct neurons, and possesses a topography which permits interaction with components of the extracellular sensillum lymph.     

Discontinuous gas exchange in the fire ant,Solenopsos invicta Buren: Caste differences and temperature effects

The discontinuous gas exchange cycle (DGC), the cyclic release of CO(2) and uptake of O(2), were investigated in workers and female and male alates of the red imported fire ant, Solenopsis invicta Buren, using real-time CO(2) emission measurement by flow-through respirometry. All S. invicta castes displayed discontinuous emission of CO(2) in the temperature range of 15-25 degrees C, but only male alates and workers exhibited a DGC at 30 degrees C. The closed (C) and flutter (F) periods of the DGC were distinguishable in alates of both sexes at the lowest temperature, but not clearly differentiated in females at temperatures above 15 degrees C, in males above 20 degrees C, or workers at any temperature. DGC frequency increased for all castes as temperature increased, ranging from a low of 0.9+/-0.05 mHz (male alates at 15 degrees C) to 18+/-0.79 mHz (workers at 30 degrees C). O period (or burst) volumes of all castes decreased as temperature increased, and increased with body mass - this mass effect was most pronounced at lower temperatures. Q(10) values for DGC frequency (4.27, 5.81, and 5.62 for workers, female and male alates, respectively) were high compared with Q(10)'s for standard Vdot;(CO(2)). Differences in the salient characteristics of the DGC between castes are presented and discussed, and S. invicta DGC patterns are compared to known values for some other ant species.     

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.     

The relative importance of intracellular proteolysis and transport on the yield of the periplasmic enzyme penicillin amidase in Escherichia coli*

Intracellular proteolysis is an important mechanism for regulating the level of the periplasmic enzyme penicillin amidase in Escherichia coli. Evidence is presented that the active enzyme is localized in the periplasmic space and maturation of pro-enzyme occurs during transport through the cytoplasmic membrane or rapidly after its entrance in the periplasm. The rate constants of the transport through cytoplasmic membrane and of the intracellular proteolysis were estimated to be 0.01 h and 0.5 h, respectively. This indicates that more than 90% of the synthesized pre-pro-enzyme is lost by intracellular proteolysis occurring in the cytoplasm.     

Hemodynamic-kinetic controlled extracorporeal circulation in heart surgery]

Mimicking the physiological characteristics of the circulatory system, pulsatile bloodflow has also been introduced into extracorporeal perfusion to avoid known postoperative complications. In a mathematical consideration of the situation bloodflow is seen as a function of time F(t) for approximately constant vessel diameter over a given time. The kinetic energy of a column of blood produced by the heart-lung machine is transmitted directly to the arterial circulation via the aorta. The nature of the energy release can give rise to both positive (organ perfusion) and negative (damage to endothelium) effects. This study investigates how this energy release can be optimised, using the following experimental approach. A Doppler flow-measuring probe is placed on the ascending aorta to monitor the extracorporeal circulation. At the same time, the blood pressure is measured and converted to a pressure-flow curve via an A/D converter. On the basis of the parameters thus obtained, the energy released by the heart-lung machine is calculated. By regulating the functional parameters of a new generation of heart-lung machines, the bloodflow can then be adapted to the physiological requirements. Within the pulse period (cycle) a 20% rise phase ending in a slightly increasing plateau is established. The energy increase within a cycle should not exceed 150 joules. To optimize the mode of functioning of the heart-lung machine, we introduced the "energy-equivalent pressure" (EEP). Adaptation of the EEP to the physiological conditions required a basic flow of 60% at a pulse rate of 60/min and a pulse duration of 35% within the pulsatile flow interval.     

Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR restriction analysis of 16S rDNA

The rhizosphere of Trifolium repens and Lolium perenne was divided into three fractions: the bulk soil, the soil adhering to the roots and the washed roots (rhizoplane and endorhizosphere). After isolation and purification of DNA from these fractions, 16S rDNA was amplified by PCR and cloned to obtain a collection of 16S rRNA genes representative of the bacterial communities of these three fractions. The genes were then characterized by PCR restriction analysis. Each different profile was used to define an operational taxonomic unit (OTU). The numbers of OTUs and the numbers of clones among these OTUs allowed to calculate a diversity index. The number of OTUs decreased as root proximity increased and a few OTUs became dominant, resulting in a lower diversity index. In the root fraction of T. repens, the restriction profile of the dominant OTU matched the theoretical profile of the 16S rRNA gene of Rhizobium leguminosarum. This study showed that plant roots create a selective environment for microbial populations.     

Patterns of Gene Duplication in Lepidopteran Pheromone Binding Proteins

We have isolated and characterized cDNAs representing two distinct pheromone binding proteins (PBPs) from the gypsy moth, Lymantria dispar. We use the L. dispar protein sequences, along with other published lepidopteran PBPs, to investigate the evolutionary relationships among genes within the PBP multigene family. Our analyses suggest that the presence of two distinct PBPs in genera representing separate moth superfamilies is the result of relatively recent, independent, gene duplication events rather than a single, ancient, duplication. We discuss this result with respect to the biochemical diversification of moth PBPs. Received: 19 March 1997 / Accepted: 11 July 1997