Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

Diversity of Siphonaria Sowerby I, 1823 (Gastropoda,Siphonariidae) in the Seychelles Bank and beyond

Molecular phylogenetic studies have shown that the characters of the reduced shell of the false limpets of the genus Siphonaria Sowerby I, 1823 are highly variable and often insufficient for species delimitation. The taxonomy and distribution of Siphonaria in the Indian Ocean are poorly known. We sampled Siphonaria in the Seychelles Bank to check the occurrence of recorded species using DNA sequences and to study the paths through which Siphonaria species have colonised the Seychelles Bank by reconstructing their phylogenetic relationships. Analyses of a dataset comprising 16 S rRNA gene sequences of 33 specimens from the Seychelles Bank and 300 additional Siphonaria sequences from other regions from GenBank with various methods for species delimitation resulted in 19–102 primary species hypotheses. Assemble Species by Automatic Partitioning provided a conservative estimate of the species number (42) in which several indisputable species were lumped. The results of Automatic Barcode Gap Discovery depended strongly on the assumed prior maximum intraspecific divergence, whereas the tree-based methods Generalised Mixed Yule Coalescent and Poisson Tree Processes resulted in high overestimates. The specimens from the Seychelles Bank represent three clades, belonging to the Siphonaria ‘atra’ group, the Siphonaria ‘normalis’ group and a possibly undescribed species recorded previously only from Hainan. At least two of the three species recorded from the Seychelles Bank came from the east, i.e., from the Coral Triangle in the Indo-Australian Archipelago, the region with the highest marine biodiversity worldwide. A major transport mechanism across the Indian Ocean was probably the South Equatorial Current.     

Proteomic fingerprinting enables quantitative biodiversity assessments of species and ontogenetic stages in Calanus congeners (Copepoda,Crustacea) from the Arctic Ocean

Species identification is pivotal in biodiversity assessments and proteomic fingerprinting by MALDI-TOF mass spectrometry has already been shown to reliably identify calanoid copepods to species level. However, MALDI-TOF data may contain more information beyond mere species identification. In this study, we investigated different ontogenetic stages (copepodids C1–C6 females) of three co-occurring Calanus species from the Arctic Fram Strait, which cannot be identified to species level based on morphological characters alone. Differentiation of the three species based on mass spectrometry data was without any error. In addition, a clear stage-specific signal was detected in all species, supported by clustering approaches as well as machine learning using Random Forest. More complex mass spectra in later ontogenetic stages as well as relative intensities of certain mass peaks were found as the main drivers of stage distinction in these species. Through a dilution series, we were able to show that this did not result from the higher amount of biomass that was used in tissue processing of the larger stages. Finally, the data were tested in a simulation for application in a real biodiversity assessment by using Random Forest for stage classification of specimens absent from the training data. This resulted in a successful stage-identification rate of almost 90%, making proteomic fingerprinting a promising tool to investigate polewards shifts of Atlantic Calanus species and, in general, to assess stage compositions in biodiversity assessments of Calanoida, which can be notoriously difficult using conventional identification methods.     

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes

 Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level. Accepted: 16 February 1996     

Molecular mechanisms of endotoxin activity

Endotoxin (lipopolysaccharide, LPS), a constitutent of the outer membrane of the cell wall of gramnegative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intra-cellular signal transduction pathways.     

Refined solution structure of human profilin I.

Profilin is a ubiquitous eukaryotic protein that binds to both cytosolic actin and the phospholipid phosphatidylinositol-4,5-bisphosphate. These dual competitive binding capabilities of profilin suggest that profilin serves as a link between the phosphatidyl inositol cycle and actin polymerization, and thus profilin may be an essential component in the signaling pathway leading to cytoskeletal rearrangement. The refined three-dimensional solution structure of human profilin I has been determined using multidimensional heteronuclear NMR spectroscopy. Twenty structures were selected to represent the solution conformational ensemble. This ensemble of structures has root-mean-square distance deviations from the mean structure of 0.58 A for the backbone atoms and 0.98 A for all non-hydrogen atoms. Comparison of the solution structure of human profilin to the crystal structure of bovine profilin reveals that, although profilin adopts essentially identical conformations in both states, the solution structure is more compact than the crystal structure. Interestingly, the regions that show the most structural diversity are located at or near the actin-binding site of profilin. We suggest that structural differences are reflective of dynamical properties of profilin that facilitate favorable interactions with actin. The global folding pattern of human profilin also closely resembles that of Acanthamoeba profilin I, reflective of the 22% sequence identity and approximately 45% sequence similarity between these two proteins.     

High-pressure, reverse-phase partition chromatograhy separation of diethylstilbestrol metabolites and analogs.

Various conjugated and oxidative metabolites of diethylstilbestrol were effectively separated by high-pressure, reverse-phase liquid chromatography on C₈- and C₁₈-hydrocarbon phases with water-methanol gradients.     

A novel fluorinated derivative of diethylstilbestrol.

The synthesis of the diethylstilbestrol (DES) derivative with fluorine atoms present in the positions ortho to the hydroxyl in each ring is described. In vitro studies in a system containing horse radish peroxidase/H2O2 demonstrate extensive oxidation of tetrafluorodiethylstilbestrol to the corresponding dienestrol derivative. Tetrafluorodiethylstilbestrol and DES had comparable in vivo uterotropic activities at a dose of 100 microgram/kg. Competitive binding experiments demonstrated 20-25 fold reduced interaction with the mouse uterine estrogen receptor. This compound may be useful as an experimental estrogen in distinguishing between the biological and toxic effects of DES.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,protein synthesis,and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of ³⁵S-sulfate and ¹⁴C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.Growth rate constants calculated from increases in cell numbers, protein, and ³⁵S and ¹⁴C in the various fractions indicated nearly balanced growth in A. luteo-violaceus, with constants derived from all biosynthetic parameters agreeing within 5% during the exponential phase. In contrast, protein synthesis and ³⁵S incorporation into residue protein constants were 30% higher than constants derived from cell counts and incorporation of ¹⁴C in P. halodurans. Therefore the cellular protein content P. halodurans varied over a two-fold range, with maximum protein per cell in the late exponential phase. A distinct reduction in the rate constants for total protein and ³⁵S incorporation into residue protein foreshadowed entry into the stationary phase more than one generation before other parameters.Incorporation of ³⁵S-sulfate into residue protein paralleled protein synthesis in both bacteria. The weight percent S in protein agreed well with the composition of an average protein derived from the literature. Sulfur incorporation into protein may be a useful measurement of marine bacterial protein synthesis.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid - CFU colony forming unit