Elevated CO2 Levels do not Affect the Shell Structure of the Bivalve Arctica islandica from the Western Baltic

Shells of the bivalve Arctica islandica are used to reconstruct paleo-environmental conditions (e.g. temperature) via biogeochemical proxies, i.e. biogenic components that are related closely to environmental parameters at the time of shell formation. Several studies have shown that proxies like element and isotope-ratios can be affected by shell growth and microstructure. Thus it is essential to evaluate the impact of changing environmental parameters such as high pCO₂ and consequent changes in carbonate chemistry on shell properties to validate these biogeochemical proxies for a wider range of environmental conditions. Growth experiments with Arctica islandica from the Western Baltic Sea kept under different pCO₂ levels (from 380 to 1120 µatm) indicate no affect of elevated pCO₂ on shell growth or crystal microstructure, indicating that A. islandica shows an adaptation to a wider range of pCO₂ levels than reported for other species. Accordingly, proxy information derived from A. islandica shells of this region contains no pCO₂ related bias.     

The Effect of Dietary Energy Restriction on Body Weight Gain and the Development of Noninsulin-Dependent Diabetes Mellitus (NIDDM) in Psammomys obesus

Food intake was restricted to 75% of ad libitum levels in 37 male Psammomys obesus (Israeli Sand Rats) from the ages of 4 (weaning) to 10 weeks. Energy restriction reduced the mean body weight at 10 weeks by 29% compared with 44 ad libitum fed controls. Hyperglycemia was prevented completely in the food-restricted group, and mean blood glucose concentrations were significantly reduced (3.8 ± 0.2 vs. 5.5 ± 0.4 μmol/L; p<0.05) compared with control animals. Plasma insulin concentrations were also decreased significantly compared with ad libitum fed controls (105 ± 13 vs. 241 ± 29 mU/L;p<0.05). Although energy restriction prevented hyperglycemia from developing in 10-week-old P. obesus, 19% of the food restricted animals still developed hyperinsu-linemia. We concluded that hyperphagia between the ages of 4 to 10 weeks may be essential for the development of noninsulin-dependent diabetes mellitus in P. obesus, but that hyperinsulinemia may still occur in the absence of hyperphagia and hyperglycemia, suggesting a significant genetic influence on the development of hyperinsulinemia in this animal model.     

The course control system of beetles walking in an air-current field

The wind-orientated walk of carrion beetles Necrophorus humator F. was analysed under closed-loop conditions with a walking compensator and under openloop conditions with a paired tread wheel (Fig. 1).

Elektronenmikroskopische Untersuchungen über die Differenzierung von Insektenmuskeln während der Metamorphose

Zusammenfassung Bildung und Entwicklung der Darmmuskulatur und der dorsoventralen und longitudinalen, indirekten Flugmuskeln der Fleischfliege Phormia regina während der Metamorphose wurden elektronenmikroskopisch untersucht. Dabei wurden in der Anlage der myofibrillären Strukturen zwischen neugebildeten dorsoventralen Flugmuskeln einerseits und transformierten Darm- und longitudinalen Flugmuskeln andererseits deutliche Unterschiede festgestellt.Beim dorsoventralen Flugmuskel lösen sich die Z-Scheibenvorläufer als elektronendichte Bezirke von den beiden Differenzierungsfronten ab, die sich gleichzeitig von der Mitte einer Faser zu deren Ansatzstellen am Außenskelet verschieben. Den transformierten Muskeln fehlen diese Fronten. Hier entstehen Z-Scheibenvorläufer zwar ebenfalls in Form dichter Bezirke, jedoch im Innern der Fasern.Zu Beginn der Wachstumsphase sind in allen drei untersuchten Muskeln Fibrillenvorläufer mit dense-body-ähnlichen Z-Elementen vorhanden. In beiden Flugmuskeltypen vergrößern sie ihren Durchmesser gleichmäßig nach allen Richtungen. Bei der Darmmuskulatur dagegen wachsen die Z-Elemente, indem nur an einer oder zwei sich gegenüberliegenden Stellen des Umfangs zusätzliches Z-Scheibenmaterial aufgelagert wird. Die Differenzierung wird hier durch die Ausbildung einer perforierten Z-Scheibe abgeschlossen, die durch Fusion zahlreicher Z-Elemente entsteht. Die bei dieser Entwicklung auftretenden Zwischenstadien werden mit glatten, schräggestreiften und quergestreiften Muskeln anderer wirbelloser Tiere verglichen und im Hinblick auf die Evolution perforierter und solider Z-Scheiben diskutiert.

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On the differentiation of insect muscles during metamorphosis

Summary The formation and development of three types of imaginai muscle fibres in the blow-fly Phormia regina were investigated by electronmicroscopy. With this technique distinct differences were found in the predisposition (anlage) of myofibrillar structures in newly formed dorsoventral flight muscles on the one side and transformed longitudinal flight muscles and visceral muscles on the other.In dorsoventral flight muscles the precursors of Z-disks were detached as dense bodies from two fronts of differentiation activity (Differenzierungsfronten) which advanced from the centre of the fibres to their ends at insertion spots on the external skeleton. In transformed muscles the precursors of Z-disks developed likewise from dense bodies. These were, however, formed in the interior of the fibre, since differenzierungsfronten were absent.At the beginning of the growth period the three types of muscles contained precursors of fibrils with Z-elements, the latter of which resembled dense bodies. In both types of flight muscles these elements grew uniformly in all directions thus forming solid Z-disks. The Z-elements of visceral muscles, on the other hand, grew by accumulating additional material merely at one spot or at two opposite spots. In these muslces the process of differentiation was completed by the fusion of numerous dense bodies which resulted in the formation of perforated Z-disks.The described intermediate stages in development were compared with the final structure in smooth, obliquely and cross-striated muscles of various invertebrates. This confrontation allows certain conclusions regarding the evolution of perforated and solid Z-disks.

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Für die Anregung zu dieser Arbeit und für wertvolle Ratschläge sowie die Durchsicht des Manuskripts bin ich Herrn Prof. Dr. E. Zebe zu außerordentlichem Dank verpflichtet.

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Auszugsweise vorgetragen auf der 61. Tagung der Deutschen Zoologischen Gesellschaft, Heidelberg 1967.

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Stipendiat der Deutschen Forschungsgemeinschaft.     

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.     

Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes

Immunotherapy of cancer could be possible in cases in which competent effector T cells can be induced. Such an approach depends on expression of tumour-specific antigens by the tumour cells and on the availability of sufficient costimulatory support for activation of cytotoxic T lymphocytes. Here, a strategy for helper T cell recruitment for induction of tumour-specific cytotoxic immune responses is presented. Allogenic MHC class II molecules were introduced into tumour cells by cell fusion. These hybrid cells, when injected into mice, induced rejection of an established tumour. The contribution of CD4-expressing helper T cells in the induction phase and of CD8-expressing T cells in the effector phase of the immune response was demonstrated. The approach described could be applicable to cases in which a suitable tumour antigen is present but not identified; it employs regulatory interactions that govern physiological immune responses and is designed to be minimally invasive.     

Information about previous phosphate fluctuations is stored via an adaptive response of the high-affinity phosphate uptake system of the cyanobacterium Anacystis nidulans

The high-affinity uptake system of phosphatelimited cyanobacterium Anacystis nidulans [Synechococcus leopoliensis (Raciborski) Komarek] is characterized by a threshold value below which uptake cannot occur. Here it is shown that, if phosphate-limited cyanobacteria are challenged with a short pulse of high phosphate concentration that appreciably exceeds this threshold value, the uptake system undergoes an adaptive response, leading to the attainment of new kinetic properties and a new threshold value. These new properties are maintained for several hours after the pulse. A notable characteristic of this new state is a wide linear dependence of the uptake rate on the external phosphate potential that is a function of the driving force of the uptake process. According to theoretical arguments it is shown that this “linear operation mode” can be explained by the simultaneous operation of several uptake systems with different, staggered threshold values and kinetic properties. Moreover, the new linear uptake properties, in turn, reflect the prehistory of phosphate supply experienced by the population. The consequences of this result with regard to environmental fluctuations of the phosphate concentration in lakes are discussed.     

INFLUENCE OF FLUCTUATING PHOSPHATE SUPPLY ON THE REGULATION OF PHOSPHATE UPTAKE BY THE BLUE-GREEN ALGA ANACYSTZS NIDULANS1

The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of ³²P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range .     

Secondary structural changes in globular protein induced by a surfactant: Fourier self-deconvolution of FT-IR spectra

Conformational changes in ovalbumin, a globular protein, induced by an anionic surfactant, sodium dodecyl sulfate (SDS), have been monitored by an FT-IR spectrometer using ZnSe cylindrical internal reflection optics which allows high quality IR spectra to be obtained in water solution. The most notable change, on addition of SDS, occurs in the composite band of the Amide I absorption band and the vibrational frequency of the composite C = O bond shifts from 1639 cm-1 to 1652 cm-1. On the other hand, the position of the Amide II band remains fairly unchanged. Comparison of the various peak positions in the deconvoluted spectra for the native protein and the perturbed protein clearly shows the effect of SDS on the secondary structures of the protein. SDS unfolds the protein. It increases the helix content slightly. More importantly, it alerts the beta sheet structure, destroying it almost completely in the Amide I region, while retaining it in its neighbourhood. In the deconvoluted spectra of the perturbed protein, a band at 1531 cm-1 indicates generation of some beta turns. We used the second derivative of the deconvoluted spectra for fixing positions of minor peaks and shoulders. The results of this study indicate that the deconvolution of the normal IR spectra, consisting of composite bands, provides evidence for the specific secondary structures in a protein and for the way they are affected by changes in the environment, e.g., the addition of SDS. This makes it possible to relate conformational changes to specific secondary structures.     

Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.