A Quantitative Microtiter Plate Nuclease Assay Based on Ethidium/DNA Fluorescence

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellularSerratia marcescensendonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.     

An RsaI polymorphism in the transcribed region of the neurofibromatosis (NF1)-gene

We describe an RsaI polymorphism in the transcribed region (exon 5) of the neurofibromatosis type 1 (NF1)-gene     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

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.     

The evolution of brood parasitism: the role of facultative parasitism

The hypothesis that facultative brood parasitism may serve asan intermediate step in the evolutionary transition from purelyparental reproduction to obligate parasitism was investigated.The population dynamics of a host-parasite complex were computer-simulatedin a model that incorporated different intensities of parasitismand host defense and considered a simplified semelparous birdspecies living in a homogeneous habitat The individuals usetwo different breeding strategies: provide parental care orparasitize the nest of those providing parental care. Underobligate parasitism, the parasites appeared unsuccessful, drovethe host population to extinction, or coexisted with the hostin stable or oscillating proportions. The behavior of the systemdepended on both the effectiveness of the parasite and the defenseof the host. Under facultative parasitism (making the best ofa bad job), the parasites reduced host numbers but did not reducethe population size below the number of breeding sites. Thus,facultative parasitism provides a better opportunity for thedevelopment of defense in the host. The population of a hostthat shows a certain level of defense can be more successfullyinvaded by obligate parasites so that stable coexistence ofhosts and parasites is possible.     

Occurrence of α-acetolactate decarboxylases among lactic acid bacteria and their utilization for maturation of beer

Summary Acetolactate decarboxylase activity has been detected among three genera, nine species and 263 strains of lactic acid bacteria tested in the course of a screening for acetolactate decarboxylases amenable for use in brewing as maturation aid. Streptococcus diacetylactis strain FD-64-D was found to generate a decarboxylase exhibiting a satisfactory activity and an excellent stability at the pH prevailing in beer and wort. This decarboxylase could not be solubilized but enzymatically active, freeze-dried cells were effective for satisfactory flavour maturation of beer although difficulties were encountered during attempts to remove the applied cell material by filtration of the beer. Lactobacillus casei DSM 2547 was likewise found to produce a decarboxylase exhibiting a satisfactory activity and stability at the low pH of beer and which, in addition, was readily solubilized. A method has been developed for pilot scale production of preparations of this decarboxylase suitable for use in brewing.Abbreviations DSM Deutsche Sammlung von Microorganismen - EDTA Ethylene diaminetetra-acetic acid     

11-Trans leukotriene C: A naturally occurring slow reacting substance

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9,11-$\text{trans}\text{-14-}\text{cis}\text{-eicosatetraenoic acid (11-}\text{trans}$ leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.     

Terminal distribution of retinal fibers in the tegu lizard (Tupinambis nigropunctatus)

Summary The retinal projections in the tegu lizard were traced using degeneration-silver methods. Bilateral projections were found to the dorsolateral geniculate and the posterodorsal nuclei. Unilateral, crossed projections were traced to the suprachiasmatic nucleus, the ventrolateral geniculate nucleus, the mesencephalic lentiform nucleus, nucleus geniculatus praetectalis, the ectomammillary nucleus, and the optic tectum. Some of these connections are distinctly different from those reported in other reptiles and suggest that important interspecific variations occur among reptiles.     

Nutrient content and dynamics in north Swedish shrub tundra areas

Forbs and leaves of deciduous shrubs had high concentrations of Ca, Mg, K, P, and N. Deciduous dwarf shrubs had intermediate concentrations but higher than evergreen dwarf shrubs. Monocots, cryptogams, woody and belowground tissues had low concentrations.
Plant nutrient concentrations and nutrient content in soil organic matter increased from dry towards moist tundra areas.
The residence time of nutrients was considerably less than ten years in surface litter, but several decades or centuries in total organic matter. The longest residence time was found in the moist part of the tundra.
N, K, and P in Betula nana leaves were translocated to a great extent prior to leaf fall, whereas Mg and particularly Ca were only slightly translocated. As on other tundra areas shortage of nutrients probably limits plant growth. In that case short supply of N and F seems most probable due to retention in litter and soil organic matter.     

Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.