Studies of a viral suppressor of RNA silencing p19-CFP fusion protein: A FRET-based probe for sensing double-stranded fluorophore tagged small RNAs

Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity.     

Calpha methylation in molecular recognition. Application to substance P and the two neurokinin-1 receptor binding sites.

Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.     

Detection of 60-hertz vertical electric fields by rats

Rats were trained to press levers to indicate the presence or absence of 60-Hz vertical electric fields at intensities from 0 to 27 kV/m (rms). The probability of detecting the field increased as the strength of the field increased. The shape of the detection curve (psychometric function) for most subjects (Ss) was similar whether the discriminative stimulus was the electric field or a tone. Two protocols were used to estimate the minimum field intensity necessary to detect the field (Reiz Limen, RL). The RL was estimated to be 13.3 kV/m (rms) when using one protocol (the staircase method) and 7.9 kV/m (rms) when using another protocol (the method of constant stimuli).     

Order amidst animalcules: the Protoctista kingdom and its undulipodiated cells

The eukaryotic microorganisms have always been studied and described in the context of Zoology (as 'tiny animals), Botany (as tiny plants), Mycology (as water molds) or Microbiology (as disease agents). Because their extent (as a group of about 200,000 species) and differences from the rest of life (worthy of Protoctista kingdom status) only recently has been recognized, their classification within the Plant and Animal kingdoms remains in scandalous disarray. Admitting this problem, this paper represents a step toward achieving nomenclatural order based on ultrastructural, biochemical and evolutionary considerations.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Protoplast and leaf explant culture of Lycopersicon cheesmanii and salt tolerance of protoplast-derived calli

In vitro manipulation of Lycopersicon cheesmanii was attempted in order to evaluate its potential to improve salt tolerance of L. esculentum following somatic hybridisation of these two species. The main study concerned three populations of the typicum form (LCS) used for tissue culture and protoplast studies. The minor halophytic form (LCT) was used only for protoplast experiments. Mother plants were sown and propagated in vitro. Shoots (2-7) were obtained from explants (leaflets, petioles, cotyledons), through calli, when 3-4 mg l^–1 BA, kinetin or 2iP were used in association with auxin. IAA (0.2 and 1 mg l^–1 was the most efficient, while 2,4-D or NAA were sometimes inhibitory according to genotype and explant. Leaf protoplasts (2-5 × 10⁷/g of fresh weight) were obtained from LCS and LCT. For LCT, protoplast division declined after five subcultures of the plantlets that had been established from seeds. Protoplast-derived calli were obtained from all populations (maximum PE 30% for LCS). However, they failed to regenerate shoots. Protoplast-derived calli involved in salt tolerance tests showed that LCS17 line was the most tolerant.     

The search for indigenous lunar organic matter

Early lunar conditions are not inconsistent with the production of large quantities of prebiological organic matter now sequestered in the lunar subsurface. The absence of substantial water and organic compounds from the superficial layers of the lunar surface may not be indicative of the composition of deeper regions.     

Synthesis of an inhibitor of glutamyl-tRNA synthetase

A specific inhibitor of glutamyl-tRNA synthetase, analog of glutamyl-adenylate, the N⁶-benzoyl-L-glutamol AMP, has been synthesized. This compound does not inhibit glutaminyl-tRNA synthetase.