In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.     

Coding of odor intensity in a steady-state deterministic model of an olfactory receptor neuron

The coding of odor intensity by an olfactory receptor neuron model was studied under steady-state stimulation. Our model neuron is an elongated cylinder consisting of the following three components: a sensory dendritic region bearing odorant receptors, a passive region consisting of proximal dendrite and cell body, and an axon. First, analytical solutions are given for the three main physiological responses: (1) odorant-dependent conductance change at the sensory dendrite based on the Michaelis-Menten model, (2) generation and spreading of the receptor potential based on a new solution of the cable equation, and (3) firing frequency based on a Lapicque model. Second, the magnitudes of these responses are analyzed as a function of odorant concentration. Their dependence on chemical, electrical, and geometrical parameters is examined. The only evident gain in magnitude results from the activation-to-conductance conversion. An optimal encoder neuron is presented that suggests that increasing the length of the sensory dendrite beyond about 0.3 space constant does not increase the magnitude of the receptor potential. Third, the sensivities of the responses are examined as functions of (1) the concentration at half-maximum response, (2) the lower and upper concentrations actually discriminated, and (3) the width of the dynamic range. The overall gain in sensitivity results entirely from the conductance-to-voltage conversion. The maximum conductance at the sensory dendrite appears to be the main tuning constant of the neuron because it determines the shift toward low concentrations and the increase in dynamic range. The dynamic range of the model cannot exceed 5.7 log units, for a sensitivity increase at low odor concentration is compensated by a sensitivity decrease at high odor concentration.     

Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.     

Spatial structure of mitochondria and ER denotes changes in cell physiology of cultured tobacco protoplasts

The structure of mitochondria and of the endoplasmic reticulum (ER) in mesophyll protoplasts and regenerated cells was studied in vivo using the dye DiOC₆(3) (3,3'-dihexyloxacarbocyanine iodide) and confocal laser scanning microscopy (CLSM). The relation to the cell's physiology was investigated using a hormone-based model system for elongation and division. The structure of the mitochondria and of their population depends on the status of the cell. In freshly isolated protoplasts small spherical mitochondria are clustered around the nucleus and the chloroplasts. During the first 4 days of culture they are transformed into long vermiform organelles which distribute evenly throughout the cytoplasm. In a medium containing only auxins, cells then enter a period of expansion. Their mitochondria retain the same structure but increase in quantity. In a medium with auxins and cytokinins cells start dividing. Their mitochondria typically become numerous and very small, and are distributed throughout the cytoplasm. Both types of organization were observed during weeks of ongoing expansion or division. The ER is always present as a network close to the cell membrane. In freshly isolated protoplasts a considerable part of the ER is clustered around the chloroplasts, the remaining part of the network has a reduced complexity and is partly broken. During subsequent protoplast culture the network grows into a complex web with fine meshes incorporating lots of plate-like structures. This is the case in elongating cells as well as in dividing cells. Finally, the ER looks similar to the structure found in epidermal cells of the intact plant.     

Influence of light,DMSO and glycerol on the hatchability ofThamnocephalus platyurus Packard cysts

Effects of two light intensities and different concentration of dimethyl sulfoxide (DMSO) and glycerol on hydrated cyst hatching inThamnocephalus platyurus were studied. A maximum of 65±6% hatching was recorded within seven days at 2500 lux continuous light regime. Hatching was at a minimum during the first two days, peaked between the third and fourth days, decreased thereafter. Hatching success was a function of duration of light exposure. Eight percent of cysts hatched in the dark, while cysts exposed to 24h light and subsequently incubated in the dark showed 27±2% hatching. Hatchability was significantly increased (23%) in 0.0375% DMSO and 0.0125% glycerol. Concentration above 0.05% DMSO and 0.025% glycerol had no or a negative influence on hatching. Since low concentrations of DMSO were non-toxic, apolar compounds like Ca²⁺ ionophore can be dissolved in DMSO to study the role of Calcium in cyst hatching.     

Food dependent color patterns inThamnocephalus platyurus Packard (Branchiopoda: Anostraca); a laboratory study

Coloration of phyllopods varies from place to place and from one life stage to another. It ranges from translucent or whitish through gray, blue, green, orange, and reddish. Here, we present experimental evidence for a food- dependent color pattern inThamnocephalus platyurus Packard. The presence or absence of the synthetic pigment trans — — carotene in a baker's yeast diet was the controlling factor. All the 24 old larvae used in the experiment were whitish in color. From day 6 until the end the experiment (day 11), 100% of the shrimps under a diet with synthetic trans — — carotene (treatment 1) exhibited a characteristic color pattern which consisted of an orange color in the cercopods, and in all theracopods; the rest of the body exhibited no particular color. In comparison, 100% of the shrimps under a diet without synthetic trans — — carotene (treatment 2) were whitish throughout the body. In females from treatment 1, the ovaries and oocytes were green-bluish, while in females from treatment 2 the ovaries and oocytes were whitish. No significant differences in survival and growth were found, except that at day 9, there was a significant difference in growth, the females with the synthetic trans — — carotene group growing faster.     

Feeding the fairy shrimp Streptocephalus (Anostraca - Crustacea) with the rotifer Anuraeopsis

Laboratory-cultured Streptocephalus torvicornis were offered 8 concentrations (from 6 to 800 ind. ml^–1) of Anuraeopsis fissa for periods of 2 h 30 min. Two size classes, small (male: 14.7 mm± 1.6, female: 15.4 mm± 1.3) and large (male: 20.0 mm±2.0, female: 23.1 mm± 1.5), of S. torvicornis were used. Functional response for large S. torvicornis (both sexes) plateaued at 400 rotifers ml^–1, while in small specimens it did so at 200 prey ml^–1. Females consumed significantly more (30%) prey than males. Large males consumed maximum 4730 rotifers h^–1, females 6560 h^–1.     

Historical biogeography and morphological differentiation ofStreptocephalus torvicornis (Waga) since the Würm III-glaciation

We argue that the Würm III glaciation eradicated possible European populations ofS. torvicornis, and that today, a reconquest of Europe takes place on two fronts.A western wave has reached the Pyrenees, an eastern one now occupies most of eastern and northern Europe. The western route probably started in the Maghreb, the eastern one in the Levant and the Ponto-Caspian. Animals in the west had to move north by crossing, at right angles, a series of east-west oriented river valleys and progressed slowly; animals in the east could move up river valleys extending north-east, and moved quickly. Italy was not occupied, becauseS.torvicornis is a warm stenotherm, and by the time the climate had warmed sufficiently for it to reach the southern shore of the mediterranean (ca 6000 BP), the gap with Italy was probably too large for a crossing. Cold-loving species (of the generaBranchipus, Chirocephalus) conversely, and could freely flow across the Central Mediterranean at low sea-levels (ca 12000 BP), and now occur in Italy (and the rest of Europe) as well as in Northern Africa.A prediction of our hypothesis is that the pioneer populations in Spain and Central Europe should have been isolated longest. Thisis tested and confirmed by their comparative morphology, and two subspecies,S. t. torvicornis andS. t. bucheti are reinstated.A gap across the Nile Valley where onlyS. rubricaudatus seems to occur, deserves further study.     

Structural modifications of oxidized insulin B-chain induced by glycerol addition

H-NMR studies of the bovine insulin S-sulfonatedB-chain are reported in H₂O/D₂O (9/1) and inglycerol-d₅ (5 M) using two-dimensional NMRspectroscopy. The first results show that the oxidizedinsulin B-chain secondary structure differs from thatof native insulin by a loss of the -helixbetween the two disulfide bridges and that theglycerol favours the structuring of the peptide.