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.     

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     

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.     

Do NOE distances contain enough information to assess the relative populations of multi-conformer structures?

Summary The feasibility of determining the relative populations of multi-conformer structures from NOE-derived distances alone is assessed. Without cross-validation of the NOE restraints, any population ratio can be refined to a similar quality of the fit. Complete cross-validation provides a less biased measure of fit and allows the estimation of the correct population ratio when used in conjunction with very tight distance restraints. With the qualitative distance restraints most commonly used in NMR structure determination, cross-validation is unsuccessful in providing the correct answer. Other experimental sources are therefore needed to determine relative populations of multi-conformer structures.To whom correspondence should be addressed.     

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.     

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Höglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as Piceirhiza nigra and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).Acid irrigation of the soil had no effect on the activity of any of the investigated enzymes, soluble protein or total N-contents irrespective of whether roots were taken from the organic layer or from the upper mineral soil. Liming, however, stimulated the activity of the three enzymes in mycorrhizas of the organic layer (O_f+O_h) whereas it had no effect on the activity of the investigated enzymes of mycorrhizas in the upper mineral soil. This effect is attributed to increased contents of soluble organic nitrogen compounds in the soil of the limed plots as compared to the unlimed plots.     

A novel approach to the analysis of the initiation of embryo development in gramineae

An in vivo model system to study the initiation of embryo development is presented. From the so-called Salmon system of wheat (alloplasmic lines with a 1BL-1RS chromosome translocation), three completely isogenic and homozygous lines were produced by selection for uniformity in about 20 selfing/backcross generations as well as between sublines of doubled haploids. The line (aestivum)-Salmon is male fertile and sexual. The lines (caudata)-Salmon and (kotschyi)-Salmon are male sterile and have a parthenogenetic capacity of about 90%. The expression of nuclear-cytoplasmic male sterility is different for the two parthenogenetic lines. The initiation of autonomous embryo development at defined developmental stages of the ovaries and the maximum degree of parthenogenesis are identical in both parthenogenetic lines as proved by the auxin test and progeny analyses. The protein patterns from ovary extracts of the three isogenic lines were identical for more than 200 spots of 2-D polyacrylamide gels, confirming their homogeneity. However, one protein (P 115.1) was found 3 days before and during anthesis only in ovaries of the parthenogenetic lines. It seems to be involved in the initiation of parthenogenesis.     

Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage

Interleek-1beta (IL-1) is a key mediator of cartilage matrix degradation in osteoarthritis and rheumatoid arthritis. It was found that the IL-1-induced suppression of glycosaminoglycan (GAG) synthesis in rat articular cartilage occurred simultaneously with the accumulation of nitrite (a metabolite of nitric oxide (NO) in aqueous milieu) in the culture medium. NO-synthase inhibitors, L-NMMA and L-NIO, inhibited both these IL-1 effects. Dexamethasone suppressed GAG synthesis additively to IL-1, but did not alter nitrite accumulation. Three NO-donors (GEA 3175, SNAP and SIN-1) also had an inhibitory effect on cartilage GAG synthesis. Therefore, it is concluded that IL-1 induced suppression of GAG synthesis in rat articular cartilage is mediated by the production of NO.