Real‐time mid‐infrared imaging of living microorganisms

The speed and efficiency of quantum cascade laser‐based mid‐infrared microspectroscopy are demonstrated using two different model organisms as examples. For the slowly moving Amoeba proteus, a quantum cascade laser is tuned over the wavelength range of 7.6 µm to 8.6 µm (wavenumbers 1320 cm^–1 and 1160 cm^–1, respectively). The recording of a hyperspectral image takes 11.3 s whereby an average signal‐to‐noise ratio of 29 is achieved. The limits of time resolution are tested by imaging the fast moving Caenorhabditis elegans at a discrete wavenumber of 1265 cm^–1. Mid‐infrared imaging is performed with the 640 × 480 pixel video graphics array (VGA) standard and at a full‐frame time resolution of 0.02 s (i.e. well above the most common frame rate standards). An average signal‐to‐noise ratio of 16 is obtained. To the best of our knowledge, these findings constitute the first mid‐infrared imaging of living organisms at VGA standard and video frame rate.

     

Trans-Generational Influence of Human Disturbances in Japanese Quail: Egg Quality Influences Male Social and Sexual Behaviour

In many bird species prenatal exposure to yolk androgens of maternal origin has been found to influence offspring behavioural phenotype. In contrast to altricial birds, far less is known about maternal effects in precocial birds. In a previous experiment we found that female quail ( Coturnix japonica ) that were not previously habituated to humans (NH) produced eggs with less androgens (testosterone, androstenedione) and more progesterone when exposed to human disturbances than females habituated to humans (H). Here, we analysed social motivation and sexual behaviour of the male offspring of NH and H females. Classical behavioural test procedures were applied including separation, runway, partner choice and female encounter tests. As chicks, offspring of the NH females spent more time far from conspecifics than offspring of the H females. As adults, the same NH males showed less crowing and courtship behaviour (ritual preening) in female encounter tests than H males. Thus, maternal environment and egg quality may be key factors in the emergence of individual variability of appetitive behaviour, such as social proximity and courtship behaviour. Human disturbance of the mother seems to have triggered trans-generational effects resulting in consistently reduced social and sexual motivation in offspring until adulthood.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

<Superscript>13</Superscript>C labeling experiments at metabolic nonstationary conditions: An exploratory study

Background  

Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of ¹³C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly.     

A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.     

Action in the axon: generation and transport of signaling endosomes

Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of 'signaling endosomes'. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.     

Endocrine signatures underlying plasticity in postembryonic development of a lower termite, Cryptotermes secundus (Kalotermitidae)

SUMMARY Wood-dwelling termites are characterized by an extremely high and unique developmental flexibility that allows workers, which are immatures, to explore all caste options. The endocrine signatures underlying this flexibility are only vaguely understood. We determined juvenile hormone (JH) and ecdysteroid hemolymph titers during postembryonic development and in terminal instars of the drywood termite Cryptotermes secundus using field and laboratory colonies. Postembryonic development is characterized by a drop in JH titers at the transition from larval (individuals without wing buds) to nymphal (individuals with wing buds) instars. JH titers were low in winged sexuals and reproducing primary reproductives (<200 pg/μl) but were by an order of magnitude higher in neotenic replacement reproductives. The unique regressive molts of termites seem to be characterized by elevated JH titers, compared with progressive or stationary molts. Ecdysteroid titers were generally low in nymphal instars and in primary reproductives (<50 pg/μl). It was only during the third and fourth nymphal instars and in winged sexuals where some individuals showed elevated ecdysteroid titers. These results are the most comprehensive endocrinological data set available for any lower termite, with the potential to serve as baseline for understanding the extreme developmental flexibility underlying the evolution of social life in termites.