A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [γ³²P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.     

Miniaturisation effects in larvae and adults of Mikado sp. (Coleoptera: Ptiliidae), one of the smallest free-living insects

We present the first morphological study of larvae and adults of Mikado sp. – one of the smallest known beetles and free-living insects (body length of adult is 390–455 μm). Morphological and developmental consequences of miniaturisation in Mikado and insects in general are discussed. We used histological sectioning, scanning electron microscopy, laser confocal microscopy and 3D-computer reconstruction. For the first time we report that according to the morphometric data of Mikado sp., at least some ptiliid beetles have three larval stages. We studied the muscular system of adults and larval stages. It is shown that ptiliid beetles have nearly the complete set of muscles found in larger staphyliniform beetles. Developmental and size dependent changes in the relative volume of different organs are addressed. All organ systems change allometrically in the development of Mikado sp. as well as in comparison with larger representatives of Ptiliidae and closely related groups of beetles, such as Staphylinidae. We conclude that the factors limiting miniaturisation are the size of the neural system, associated with the number and size of neurons, the mass of the skeleton, the egg size (free-living insects), and consequently the volume of the reproductive system.     

A dynamic programming approach for finding common patterns in RNAs.

We developed a dynamic programming approach of computing common sequence structure patterns among two RNAs given their primary sequences and their secondary structures. Common patterns between two RNAs are defined to share the same local sequential and structural properties. The locality is based on the connections of nucleotides given by their phosphodiester and hydrogen bonds. The idea of interpreting secondary structures as chains of structure elements leads us to develop an efficient dynamic programming approach in time O(nm) and space O(nm), where n and m are the lengths of the RNAs. The biological motivation is given by detecting common, local regions of RNAs, although they do not necessarily share global sequential and structural properties. This might happen if RNAs fold into different structures but share a lot of local, stable regions. Here, we illustrate our algorithm on Hepatitis C virus internal ribosome entry sites. Our method is useful for detecting and describing local motifs as well. An implementation in C++ is available and can be obtained by contacting one of the authors.     

Protoxenidae fam. nov. (Insecta, Strepsiptera) from Baltic amber — a 'missing link' in strepsipteran phylogeny

A male specimen of a new strepsipteran genus and species ( Protoxenos janzeni gen. et sp. nov.) and family (Protoxenidae fam. nov.) found in Baltic amber is described and illustrated. It shows features which are apparently more plesiomorphic than in hitherto known strepsipterans, such as laterally inserted eight-segmented antennae, very robust mandibles with a broad base, a prominent galea, a comparatively short, transverse metapostnotum, hindwings that are feebly extended in a rostrocaudal direction, and equally sclerotized abdominal tergites and sternites. Based on a cladistic analysis of 46 characters of males of 11 genera and three outgroup taxa, P. janzeni is the sister group of all other known strepsipterans, and Mengea the sister group of Strepsiptera s.s . Eoxenos is the sister group of the remaining extant strepsipterans and Mengenillidae is therefore paraphyletic. Newly established groundplan features of Strepsiptera will facilitate the clarification of the systematic position of the Order in future studies.     

The ITS region as a target for characterization of fungal communities using emerging sequencing technologies

In a previous work, it was observed that the swarming of polyamine-deficient Proteus mirabilis ( speB :: sm ) was severely inhibited on Luria–Bertani (LB) swarming plates (LBSw) (LB, 0.5% glucose, 0.5% agar), and it was clarified that extracellular putrescine was important as a signaling molecule for the induction of swarming in P. mirabilis . However, a polyamine-deficient strain (delta- speAB delta- speC ) of Escherichia coli swarmed as well as the parental strain on LBSw plates. We report that the swarming phenotype of a polyamine-deficient E. coli strain is dependent on spermidine and PotABCD, a spermidine importer.     

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The peptides include bradykinin, Hyp³-bradykinin, des-Arg⁹-bradykinin and fragments of fibrinogen α-chain (Fib-α_[605–629]), inter-α-trypsin inhibitor heavy chain 4 (ITIH_4[666–687]) and complement component 4a (C4a_{[1337–1350]}). Ile¹³-ITIH_4[666–687], d20-C4a_{[1337–1350]} and Sar-D-Phe⁸-des-Arg⁹-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM d-l-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10–500 ng/ml), Hyp³-bradykinin (4–200 ng/ml), des-Arg⁹-bradykinin (2–100 ng/ml), Fib-α_[605–629] (120–3000 ng/ml), ITIH_4[666–687] (0.4–10 ng/ml) and C4a_{[1337–1350]} (1–25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.     

MR contrast agent composed of cholesterol and peptide nucleic acids: Design,synthesis and cellular uptake

A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5 μM). However, after uptake into cells the agent was located predominantly in endosomes like a similar cell penetrating peptide conjugated probe. Our results indicate that this newly developed contrast agent could be used for the labeling of cells for optical as well as magnetic resonance imaging.     

Concentration of live pico- and nanoplankton by means of tangential flow filtration

A tangential flow filtration system for concentrating live pico-and nanoplankton was tested on natural seawater (<20 µm)from Kiel Bight. Twenty- to thirty-fold concentration of samplesranging from 79 to 152 dm³ was easily achieved, the time necessarydepending on sample volume, pumping pressure and filtrate/retentateratio. Losses of different kinds of cells in relation to theconcentration factor were quantified. Recovery rates were negativelycorrelated with the frequency of sample recirculation throughthe filtration unit.