Effects of energy deprivation on the fly pupil mechanism: evidence for a rigor state

The energy dependence of the pupil pigment-migrations in the fly Musca domestica was studied in live animals, using optical techniques and nitrogen-gas induced anoxia. The results obtained can be summarized in 3 points:

1. Energy deficiency can make the pupil mechanism stop in any state, extreme or intermediate.
2. Anoxia induced during intermittent stimulation makes the pupil stop in the closed state (aggregated pigment granules).
3. During long-term anoxia the pupil very slowly opens (dispersal of pigment granules), irrespective of ambient intensity.

The slow anoxic opening (point 3) is more than 1000 times slower than that predicted for free diffusion of pigment granules in water. Assuming realistic values of cytoplasm viscosity, this implies that anoxia causes the pigment granules to attach to rigid structures in the cells, in analogy with the rigor state in anoxic muscles. The rigor phenomenon in the pupil mechanism prevents experimental discrimination between active and passive processes of pigment migration. Normal pupil opening has a time course which agrees reasonably with a passive diffusion process, but it is argued that an active transportation of granules away from the rhabdom is more likely in the dark adapted eye.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates. Correspondence to: M. Rättö     

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

The Effect of Transport Stress on Plasma Levels of Catecholamines,Cortisol, Corticosteroid-Binding Globulin,Blood Cell Count,and Lymphocyte Proliferation in Pigs

The effect of transport stress on the plasma levels of catecholamines, Cortisol, and corticosteroid-binding globulin were studied in 6 gilts. To assess the effect on immune status, white blood cells were also counted and the cell-mediated immunity was estimated. The adrenaline level increased significantly during transport, from a basal mean level of 0.03 ng/ml to a plateau level of 0.11 to 0.12 ng/ml. The noradrenaline level fluctuated, but not constantly, during transport. The mean plasma Cortisol level before loading was approximately 40 nmol/1 and rose immediately after the start of transport to 70 nmol/1 (p< 0.05) and to 87 nmol/1 (p< 0.01) within 10 and 30 min, respectively. After unloading the Cortisol level rapidly decreased and a minimum level was seen 4 h after the transport, whereafter the diurnal rhythm was resumed. The plasma corticosteroid-binding globulin level increased nonsignificantly during the day of transport, from 25 nmol/1 to a level of 34 nmol/1, and it continued to increase until a plateau level was reached on the second day after transportation. The total white blood cell number increased significantly (from 13.7 to 15.5 × 109 cells/1), the number of lymphocytes decreased significantly (from 8.4 to 7.0 × 109 cells/1), and the number of polymorphonuclear neutrophils increased significantly (from 4.3 to 7.2 × 109 cells/1) during transport. No significant variation in the proliferation response was seen in the whole blood cell cultures. The main results were the significant signs of simultaneous activity of both the adrenal cortex and the adrenal medulla during transport.     

Automatic control of the extra-corporal bypass: system analysis, modelling and evaluation of different control modes.

Automatic control of the blood gas parameters during extracorporeal circulation has the potential to improve the quality of this procedure and to relieve the personnel from a time consuming task. This paper describes a model of the underlying system for a standard clinical set-up and pinpoints the major difficulties which are the variations of the process gains and the blood- and gas-flow dependent dead times and time constants. Scheduled PI-controllers both for the arterial oxygen as well as for the carbon dioxide partial pressure were designed. Scheduling was based on the blood flow rate. These controllers were tested in a simulation environment. The control systems remained stable under all tested operating condition, but if the blood flow rate was changed abruptly rather large load errors occurred. The performance was improved markedly by adding a feed-forward control path which directly influences the actuating signals based on the actual blood flow rate and the hemoglobin contents, variables which are measured anyway. The major conclusion of this study is to use such direct feed-forward compensation even if more sophisticated control algorithms are used.     

CU(II)-catalyzed oxidation of Alzheimer's disease beta-amyloid peptide and related sequences: remarkably different selectivities of neurotoxic betaAP1-40 and non-toxic betaAP40-1.

We investigated the CuII-catalyzed oxidation of beta-amyloid peptides betaAP10-20 and betaAP40-1 by tandem mass spectrometry and compared oxidation yields and selectivities to those for betaAP1-16, betaAP1-28 and betaAP1-40, which were obtained earlier (26). While betaAP1-16, betaAP1-28 and betaAP1-40 showed an almost exclusive oxidation of His residues to 2-oxo-histidine, the selectivity pattern is changed for betaAP10-20,which shows oxidation of His but also hydroxylation of Tyr and Phe. In contrast to betaAP1-40, the reverse sequence betaAP40-1 shows a strong selectivity for the hydroxylation of Tyr31 while only negligible His oxidation is observed at early time points. These selectivity patterns show the importance of the geometry of the metal-binding site for peptide/protein oxidation. The significantly different characteristic of betaAP1-40 and betaAP40-1 with regard to metal catalyzed processes may be related to the differences in the neurotoxic properties of these sequences.     

EPR and multi-field magnetisation of reduced forms of the binuclear iron centre in ribonucleotide reductase from mouse

Mouse ribonucleotide reductase is composed of a 1?:?1 complex of two homodimeric subunits and catalyses the first unique step on the biochemical pathway to DNA synthesis. The small subunit, protein R2, contains dinuclear iron-oxygen clusters and a tyrosyl free radical required for catalytic activity. We have studied the mixed valent and fully reduced forms of the diiron oxygen cluster from mouse R2 protein by low-temperature EPR. EPR signals of the mixed-valent states of proteins R2 reconstituted with ferrous iron and oxygen in normal and deuterated water, using the same buffers, show apparent g values of 1.92, 1.73, and 1.60 for the mixed-valent state in H₂O and 1.93, 1.73, and 1.62 in D₂O. These g values are typical for diiron-oxygen proteins, while the effect of D₂O is unprecedented for this class of proteins. We estimate the coupling constant J for the Heisenberg exchange (H?=?2J*S₁*S₂) to be J?=?–7.5±1?cm^–1 for the mixed-valent form. The diferrous R2 protein shows an integer spin EPR signal in the presence of azide or 20% glycerol. Variable temperature variable field saturation magnetisation measurements show that only in the azide-complexed R2 protein does a weak ferromagnetic coupling occur (J?=?0.26±0.05?cm^–1), while R2 protein in the absence or presence of 20% glycerol contains non-coupled mononuclear ferrous iron (S?=?2) sites.     

Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the midbrain anlage in the neural plate

her5 encodes a basic helix-loop-helix (bHLH) protein with all features characteristic of the Drosophila hairy-E(spl) family. her5 is expressed in a band of cells within the neural anlage from about 90% epiboly on to at least 36 h postfertilization (hpf). After completion of brain morphogenesis, her5-expressing cells are located in the caudal region of the midbrain, at the boundary with the rhombencephalon. Labelling of cells within the her5 expression domain in the neural plate by injection of fluorescein-dextran allows their labelled progeny to be localized in the 36-hpf-old embryo using an anti-fluorescein antibody. This shows that the her5 expression domain corresponds to the midbrain primordium, including both the tectum and the tegmentum, in the neural plate. A possible function for her5 in regionalization of the brain and/or control of the midbrain-hindbrain boundary is discussed.     

The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.