A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.     

Physico-chemical characterisation of sago starch

The physico-chemical characteristics of various sago starch samples from South East Asia were determined and compared to starches from other sources. X-ray diffraction studies showed that all the sago starches exhibited a C-type diffraction pattern. Scanning electron microscopy showed that they consist of oval granules with an average diameter around 30 μm. Proximate composition studies showed that the moisture content in the sago samples varied between 10.6% and 20.0%, ash between 0.06% and 0.43%, crude fat between 0.10% and 0.13%, fiber between 0.26% and 0.32% and crude protein between 0.19% and 0.25%. The amylose content varied between 24% and 31%. The percentage of amylose obtained by colourimetric determination agreed well with the values obtained by fractionation procedures and potentiometric titration. Intrinsic viscosities and weight average molecular weight were determined in 1M KOH. Intrinsic viscosity for amylose from sago starches varied between 310 and 460 ml/g while for amylopectin the values varied between 210 and 250 ml/g. The molecular weight for amylose was found to be in the range of 1.41×10⁶ to 2.23×10⁶ while for amylopectin it was in the range of 6.70×10⁶ to 9.23×10⁶. The gelatinisation temperature for the sago starches studied varied between 69.4°C and 70.1°C. The exponent ‘a’ in the Mark–Houwink equation and the exponent ‘’ in the equation R_g=kM was found to be 0.80 and 0.58, respectively for amylose separated from sago starch and these are indicative of a random coil conformation. Two types of pasting properties were observed. The first was characterised by a maximum consistency immediately followed by sharp decrease in consistency while the second type was characterised by a plateau when the maximum consistency was reached.     

The ephrin receptor EphB2 regulates the connectivity and activity of enteric neurons

Highly organized circuits of enteric neurons are required for the regulation of gastrointestinal functions, such as peristaltism or migrating motor complex. However, the factors and molecular mechanisms that regulate the connectivity of enteric neurons and their assembly into functional neuronal networks are largely unknown. A better understanding of the mechanisms by which neurotrophic factors regulate this enteric neuron circuitry is paramount to understanding enteric nervous system (ENS) physiology. EphB2, a receptor tyrosine kinase, is essential for neuronal connectivity and plasticity in the brain, but so far its presence and function in the ENS remain largely unexplored. Here we report that EphB2 is expressed preferentially by enteric neurons relative to glial cells throughout the gut in rats. We show that in primary enteric neurons, activation of EphB2 by its natural ligand ephrinB2 engages ERK signaling pathways. Long-term activation with ephrinB2 decreases EphB2 expression and reduces molecular and functional connectivity in enteric neurons without affecting neuronal density, ganglionic fiber bundles, or overall neuronal morphology. This is highlighted by a loss of neuronal plasticity markers such as synapsin I, PSD95, and synaptophysin, and a decrease of spontaneous miniature synaptic currents. Together, these data identify a critical role for EphB2 in the ENS and reveal a unique EphB2-mediated molecular program of synapse regulation in enteric neurons.     

Classical conditioning of breathing pattern after two acquisition trials in 2-day-old mice.

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. We hypothesized that sensory stimuli signaling the resumption of maternal care after separation may trigger an arousal and/or orienting response accompanied with concomitant respiratory changes. We performed a conditioning experiment in 2-day-old mice by using an odor (lemon) as the conditioned stimulus (CS) and maternal care after 1 h without the mother as the unconditioned stimulus (US). Each pup underwent two acquisition trials, in which the CS was presented immediately before (experimental paired group, n = 30) or 30 min before (control unpaired group, n = 30) contact with the mother. Conditioning was tested by using noninvasive whole body plethysmography to measure the respiratory response to the CS for 1 min. We found significantly stronger respiratory responses to the CS in the experimental group than in the control group. In contrast, somatomotor activity did not differ significantly between groups. Our results confirm the sensitivity of breathing to conditioning and indirectly support the hypothesis that learned feedforward processes may complement feedback pathways during postnatal maturation of respiratory control.     

Blitzkrieg in a Marine Invasion: Caulerpa racemosa var. cylindracea (Bryopsidales, Chlorophyta) Reaches the Canary Islands (North-East Atlantic)

On the basis of morphological and genetic studies (rDNA ITS1, 5.8S, ITS2, and a 18S rDNA intron), we confirm here that Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman et Boudouresque, a southwestern Australian taxon recently introduced into the Mediterranean Sea also occurs in the Canary Islands. This is the first report of C. racemosa var. cylindracea in the Atlantic. It was observed for the first time in the Canary Archipelago in 1997–1998. The speed and regional scale of expansion (north Atlantic Ocean and the Mediterranean Sea) of this invasive species appear to be among the most dramatic ever recorded. The possible outcome of this introduction in the Atlantic is discussed.     

Cumulative Risk of Bovine Mastitis Treatments in Denmark,Finland, Norway and Sweden

Data from the national dairy cow recording systems during 1997 were used to calculate lactation-specific cumulative risk of mastitis treatments and cumulative risk of removal from the herds in Denmark, Finland Norway and Sweden. Sweden had the lowest risk of recorded mastitis treatments during 305 days of lactation and Norway had the highest risk. The incidence risk of recorded mastitis treatments during 305 days of lactation in Denmark, Finland, Norway and Sweden was 0.177, 0.139, 0.215 and 0.127 for first parity cows and 0.228, 0.215, 0.358 and 0.204 for parities higher than three, respectively. The risk of a first parity cow being treated for mastitis was almost 3 times higher at calving in Norway than in Sweden. The period with the highest risk for mastitis treatments was from 2 days before calving until 14 days after calving and the highest risk for removal was from calving to 10 days after calving in all countries.The study clearly demonstrated differences in bovine mastitis treatment patterns among the Nordic countries. The most important findings were the differences in treatment risks during different lactations within each country, as well as differences in strategies with respect to the time during lactation mastitis was treated.     

Intermittent hypoxia induces transient arousal delay in newborn mice.

Previous studies suggested that defective arousal might be a major mechanism in sleep-disordered breathing such as sudden infant death syndrome and obstructive sleep apnea. In this study, we examined the effects of intermittent hypoxia (IH) on the arousal response to hypoxia in 4-day-old mice. We hypothesized that IH would increase arousal latency, as previously reported in other species, and we measured the concomitant changes in ventilation to shed light on the relationship between breathing and arousal. Arousal was scored according to behavioral criteria. Breathing variables were measured noninvasively by use of whole-body flow plethysmography. In the hypoxic group (n = 14), the pups were exposed to 5% O(2) in N(2) for 3 min and returned to air for 6 min. This test was repeated eight times. The normoxic mice (n = 14) were constantly exposed to normoxia. The hypoxic mice showed a 60% increase in arousal latency (P < 0.0001). Normoxic controls showed virtually no arousals. IH depressed normoxic ventilation below baseline prehypoxic levels, while preserving the ventilatory response to hypoxia. The breathing pattern and arousal responses recovered fully after 2 h of normoxia. We conclude that IH rapidly and reversibly depressed breathing and delayed arousal in newborn mice. Both effects may be due to hypoxia-induced release of inhibitory neurotransmitters acting concomitantly on both functions.     

Basophils and exercise-induced hypoxemia in extreme athletes.

This study examined whether the increase in histamine release (%H, i.e., plasma histamine expressed as a percentage of whole blood histamine) associated with exercise-induced hypoxemia (EIH) is related to high training-induced changes in basophil and osmolarity factors in arterial blood. All parameters were measured in 20 endurance athletes, 11 of whom presented an EIH (HT(hyp)) and 9 of whom were nonhypoxemic (HT(nor)), and in 10 untrained control subjects (UT). Measurements were made at rest, at the maximal workload of an incremental exhaustive exercise test, and at the fifth minute of recovery. %H increased during exercise in HT(hyp) (P < 0.01) but did not increase significantly in HT(nor) and UT controls. The results indicated that 1) osmolarity and Na(+) and K(+) concentrations did not differ between the two trained groups and 2) the basophil count and basophil histamine content did not differ among groups. We concluded that the %H increase associated with EIH was not due to a training effect on these parameters. The relatively low increase in histamine content during exercise in HT(hyp) in comparison to HT(nor) (P < 0.05) and UT (P < 0.01) and the low recovery vs. resting basophil count only in HT(hyp) (P < 0.01) suggested an accentuated exercise-induced basophil degranulation in the hypoxemic athletes.     

Transfomration of arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid by mouse peritoneal macrophages.

Mouse peritoneal macrophages were incubated at 37 degrees C for 30 min with arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid). Oxygenation of arachidonic acid in mouse peritoneal macrophages occurs by two major pathways: fatty acid cyclooxygenase and lipoxygenase. The major metabolite of the latter is 12-hydroxy-5,8,10,14-eicosatetraenoic acid which was identified by gas liquid chromatography on high resolution glass capillary column and mass spectrometry.     

Circular dichroism and UV melting studies on formation of an intramolecular triplex containing parallel T*A:T and G*G:C triplets: netropsin complexation with the triplex.

We have used circular dichroism and UV absorption spectroscopy to characterize the formation and melting behaviour of an intramolecular DNA triple helix containing parallel T*A:T and G*G:C triplets. Our approach to induce and to stabilize a parallel triplex involves the oligonucleotide 5'-d(G4A4G4[T4]C4T4C4-[T4]G4T4G4) ([T4] represents a stretch of four thymine residues). In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we have shown the following significant results. (i) While in the absence of MgCl2 this oligonucleotide adopts an intramolecular hairpin duplex structure prolonged by the single strand extremity 5'-d([T4]G4T4G4), the presence of millimolar concentrations of MgCl2generates an intramolecular triplex (via double hairpin formation). (ii) In contrast to the antiparallel triplex formed by the oligonucleotide 5'-d(G4T4G4[T4]G4A4G4[T4]C4T4C4), the parallel triplex melts in a biphasic manner (a triplex to duplex transition followed by a duplex to coil transition) and is less stable than the antiparallel one. The enthalpy change associated with triplex formation (-37 kcal/mol) is approximately half that of duplex formation (-81 kcal/mol). (iii) The parallel triple helix is disrupted by increasing the concentration of KCl(>10 mM), whereas, under the same conditions, the antiparallel triplex remains stable. (iv) Netropsin, a natural DNA minor groove-binding ligand, binds to the central site A4/T4of the duplex or triplex in an equimolar stoichiometry. Its association constant K is smaller for the parallel triplex ( approximately 1 x 10(7) M-1) than for the antiparallel one ( approximately 1 x 10(8) M-1). In contrast to the antiparallel structure, netropsin binding has no apparent effect on thermal stability of the parallel triple helix.