Differential regulation of two ABA-inducible genes from Craterostigma plantagineum in transgenic Arabidopsis plants

In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the -glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter.     

The Friedreich Ataxia Critical Region Spans A 150-kb Interval on Chromosome 9q13

By analysis of crossovers in key recombinant families and by homozygosity analysis of inbred families, the Friedreich ataxia (FRDA) locus was localized in a 300-kb interval between the X104 gene and the microsatellite marker FR8 (D9S888). By homology searches of the sequence databases, we identified X104 as the human tight junction protein ZO-2 gene. We generated a largescale physical map of the FRDA region by pulsed-field gel electrophoresis analysis of genomic DNA and of three YAC clones derived from different libraries, and we constructed an uninterrupted cosmid contig spanning the FRDA locus. The cAMP-dependent protein kinase γ-catalytic subunit gene was identified within the critical FRDA interval, but it was excluded as candidate because of its biological properties and because of lack of mutations in FRDA patients. Six new polymorphic markers were isolated between FR2 (D9S886) and FR8 (D9S888), which were used for homozygosity analysis in a family in which parents of an affected child are distantly related. An ancient recombination involving the centromeric FRDA flanking markers had been previously demonstrated in this family. Homozygosity analysis indicated that the FRDA gene is localized in the telomeric 150 kb of the FR2-FR8 interval.     

Confocal scanning laser microscopy of the follicular epithelium in ovarioles of the stick insect Carausius morosus

Ovarian follicles of the stick insect Carausius morosus were analyzed by confocal laser microscopy and immunocytochemistry with a view to studying cell polarity in the follicular epithelium. Such probes as anti-α-tubulin antibodies and Rh-phalloidin were employed to establish how the follicle cell cytoskeleton changes during ovarian development. Data show that α-tubulin prevails over the basal end, while F-actin appears more abundant along the apical end of the follicle cells. This finding was further corroborated by immunogold cytochemistry, showing that label along the basal end is primarily associated with microtubules, while that along the apical end is due to follicle cell microvilli interdigitating with the oocyte plasma membrane. A monoclonal antibody specifically raised against a vitellin polypeptide was used to investigate the role the follicular epithelium might play in relation to vitellogenin (Vg) uptake by the oocyte. Data show that under these conditions label is restricted to the intercellular channels of the follicular epithelium, thus providing further support to the notion that Vg enters the oocyte through the extracellular pathway leading from the basement lamina to the oocyte surface. By contrast, the use of a monoclonal antibody raised against a fat-body-derived protein of 85 kDa that is specifically sulfated within the follicle cells provides evidence for the existence of an alternative way of gaining access to the oocyte surface, that is by transcytosis through the follicular cell epithelium. These findings confirm our earlier observations on stick insect ovarioles whereby polarization in the follicular epithelium is primarily addressed to sustain a transcytotic vesicular traffic between opposite poles of the follicle cell of Vg toward the oocyte surface.     

Ultrastructural, morphometrical and immunocytochemical analyses of the exocrine pancreas in a hibernating dormouse

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in α-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of α-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.     

δ13C variability of benthic algae: effects of water colour via modulation by stream current

1. Increased water motion is expected to reduce boundary layer diffusion resistance of autotrophs, thereby enabling greater isotopic discrimination against ¹³C such that lower δ¹³C values (ratio of ¹³C : ¹²C) should ensue. A field test of this hypothesis was undertaken by sampling benthic algae in streams of differing current speed.
2. Contrary to the expected negative relationship between δ¹³C and water motion, filamentous benthic algae were found to exhibit higher δ¹³C values in rapid water.
3. Under conditions of low current in the streams studied, concentrations of dissolved organic carbon as measured by water colour are elevated through the microbial decomposition of largely terrestrial organic matter. Photoassimilation of this respired carbon by benthic filamentous algae generates ¹³C‐depletion and lower δ¹³C values, and appears to be substantial enough in the streams used in the present study to override the competing influence of water motion on boundary layer thickness.     

Antisense strategy unravels tau proteins as molecular risk factors for glutamate-induced neurodegeneration

Summary 1. We investigated the possible involvement of tau proteins in the neurotoxic process activated by glutamate using the oligonucleotide antisense strategy.2. We found that pretreatment of granule cells with an antisense oligonucleotide of the tau gene completely prevented the increase in tau immunoreactivity induced by glutamate.3. A significant amount of the tau antisense oligonucleotide (about 1 to 2% of total) was taken up by the cells and remained stable in the cells for at least 60 min. A dose-response study revealed that 25µM tau antisense oligonucleotide was the most efficacious concentration in terms of prevention of glutamate-induced tau immunoreactivity increases, without affecting basal tau expression. Higher concentrations of tau oligonucleotide antisense reduced tau immunoreactivity in control cells.4. Significantly, the concentration-response curve of glutamate for inducing neuronal death in cells pretreated with tau antisense oligonucleotide showed a shift to the right compared to those obtained in untreated or tau sense oligonucleotide-treated cells.5. Since inhibition of tau synthesis does not completely prevent but only decreases the neuronal sensitivity to glutamate, it is tempting to speculate that accumulation of tau within the neuron in response to glutamate represents one of the molecular risk factors lowering the safety margin of neurons to excitotoxic-induced injury.     

Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience.

OBJECTIVE--To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis. DESIGN--Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (delta F508, delta I506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus or family history of cystic fibrosis. SETTING--South Australian Neonatal Screening Programme, Adelaide. SUBJECTS--All 88,752 neonates born in South Australia between December 1989 and December 1993. INTERVENTIONS--Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. MAIN OUTCOME MEASURES--Identification of all children with cystic fibrosis in the screened population. RESULTS--Of 1004 (1.13%) neonates with immunoreactive trypsinogen > or = 99th centile, 912 (90.8%) had no identifiable mutation. 23 neonates were homozygotes or compound heterozygotes; 69 carried one identifiable mutation, of whom six had positive sweat tests. Median age at clinical assessment for the 29 neonates with cystic fibrosis was 3 weeks; six had meconium ileus and two had affected siblings. 63 neonates were identified as carriers of a cystic fibrosis mutation. Extra laboratory costs for measuring immunoreactive trypsinogen and direct gene analysis were $A1.50 per neonate screened. CONCLUSION--This strategy results in early and accurate diagnosis of cystic fibrosis and performs better than screening strategies based on immunoreactive trypsinogen measurement alone.     

Localization of calcium in the cyanobiont and gonidial zone ofCycas revoluta Thunb. by microelectrodes,chlorotetracycline, electron spectroscopic imaging and electron energy loss spectroscopy

Summary Ionic calcium concentration was measured in the gonidial zone of fresh coralloid roots by means of calcium microelectrodes. It was 10⁻⁶ M in the apical segments of coralloid roots and increased to 10⁻⁵ M in the gonidial zones of median and basal segments. Loosely membrane-bound calcium was evidenced by using chlorotetracycline (CTC) or ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and CTC, in cell walls of columnar cells ofCycas and in the cytoplasm of cyanobiont. Sub-cellular localization of calcium was obtained by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) analyses applied at transmission electron microscopy on thin, unstained sections of gonidial zone of coralloid roots. By means of these techniques, bound-calcium was detected inside the mucilage of apical and median segments whereas, in the basal segments, it was completely absent. In the heterocysts of apical segments of coralloid, calcium was localized on the envelope, cell walls, thylakoids and cyanophycin granules. In the gonidial zone of the basal segments, dead or degenerating heterocysts completely lacked calcium. Therefore, the high ionic calcium amounts detected in the gonidial zone of median and basal segments could represent a minor calcium uptake by the cells or release by lysed ones. The decreases in nitrogenase activity recorded in the median and basal segments of the coralloid roots paralleled the decrease in calcium amount in heterocyst envelope.