Characterization and genetic analysis of a lettuce (Lactuca sativa L.) mutant,weary, that exhibits reduced gravitropic response in hypocotyls and inflorescence stems

A lettuce (Lactuca sativa L.) mutant that exhibits a procumbent growth habit was identified and characterized. In two wild type (WT) genetic backgrounds, segregation patterns revealed that the mutant phenotype was controlled by a recessive allele at a single locus, which was designated weary. Hypocotyls and inflorescence stems of plants homozygous for the weary allele exhibited reduced gravitropic responses compared with WT plants, but roots exhibited normal gravitropism. Microscopic analysis revealed differences in the radial distribution of amyloplasts in hypocotyl and inflorescence stem cells of weary and WT plants. Amyloplasts occurred in a single layer of endodermal cells in WT hypocotyls and inflorescence stems. By contrast, amyloplasts were observed in several layers of cortical cells in weary hypocotyls, and weary inflorescence stem cells lacked amyloplasts entirely. These results are consistent with the proposed role of sedimenting amyloplasts in shoot gravitropism of higher plants. The phenotype associated with the weary mutant is similar to that described for the Arabidopsis mutant sgr1/scr, which is defective in radial patterning and gravitropism.     

Divergent roles of GSK3 and CDK5 in APP processing

Glycogen synthase kinase-3 (GSK3) and cyclin-dependent kinase 5 (CDK5) are related serine/threonine kinases that have been well studied for their role in tau hyperphosphorylation, however, little is known about their significance in APP processing. Here we report that GSK3 and CDK5 are involved in APP processing in a divergent manner. Specific inhibition of cellular GSK3 by lithium or GSK3beta antisense elicits a reduction in Abeta. Conversely, negative modulation of cellular CDK5 activity by CDK5 inhibitor, roscovitine, or CDK5 antisense stimulates Abeta production. Neither GSK3 nor CDK5 inhibition by these means significantly affected cellular APP levels or APP maturation. Moreover, oral administration of lithium significantly reduces Abeta production whereas direct ICV administration of roscovitine augmented Abeta production in the brains of PDAPP (APP(V717F)) mice. Our data support a function for both GSK3 and CDK5 in APP processing, further implicating these two kinases in the pathogenesis of Alzheimer's disease.     

Inhibition of Abeta production and APP maturation by a specific PKA inhibitor

Alzheimer's disease is characterized pathologically by extracellular amyloid beta protein (Abeta) deposition in the brain. The Abeta peptide, a 39-42 amino acid fragment, is derived from defined proteolysis of the amyloid precursor protein (APP) [Glenner et al., Appl. Pathol. 2 (1984) 357-369; Selkoe, Neuron 6 (1991) 487-498] and is the primary component of senile plaques. Although it is known that intracellular APP is subjected to posttranslational modification, the molecular mechanism that regulates the APP processing is not completely clear. In the present study, we demonstrates that H89, a specific inhibitor for cAMP dependent protein kinase A (PKA), inhibits Abeta production and APP secretion in a dose dependent manner in cells stably transfected with human APP bearing a 'Swedish mutation'. Concurrent with the effect, H89 inhibits C-terminal fragment of the APP. We also found that the PKA inhibitor abolishes the mature form of intracellular APP and accumulates the immature form. Finally, direct administration of H89 into brains of transgenic mice overexpressing human APP shows that the compound inhibits Abeta production in the hippocampal region. Our data suggests that PKA plays an important role in the maturation of APP associated with APP processing.     

Phased infectivity in Heterorhabditis megidis: the effects of infection density in the parental host and filial generation

Entomopathogenic nematodes can develop through two or more generations in the cadavers of killed insect hosts. Non-feeding infective juveniles from each generation emerge and may spend prolonged periods searching for a new host. The infectivity of the infective juveniles of Heterorhabditis megidis varies with time after emergence and may not reach a maximum until several weeks have passed. 'Phased infectivity' hypotheses propose that this pattern is adaptive, tending to reduce competition in new hosts. Here we provide further evidence that infectivity is phased in H. megidis. In addition, we show that the basic pattern is modified by infection density in the parental host and by filial generation. Two general patterns were observed: first, infective juveniles that developed under the least crowded conditions (F(1) infective juveniles produced in hosts infected with 16 parent nematodes) reached maximum infectivity after only 15 days, compared to 27 or 39 days for infective juveniles that developed under more crowded conditions (F(1) produced in hosts infected with 103 or 424 parent nematodes or F(2) infective juveniles). Second, infective juveniles had lower infectivity overall when produced under the most crowded conditions (F(2) versus F(1); highest versus lowest infection density). We propose that while lower overall infectivity is a necessary consequence of limited resource availability during infective juvenile development, the difference in the timing of peak infectivity reflects a shift in the fitness gains associated with being maximally infective either 'early' or 'late'. F(1) infective juveniles emerge several days before F(2) infective juveniles, and we suggest that filial generation and infection density in the parental host function as indicators of the potential risk of competition within new hosts.     

Inhibition of chitin synthetase from Saccharomyces cerevisiae by a new UDP-GlcNAc analogue

The synthesis and biological evaluation of a new UDP-GlcNAc competitor (I), designed to mimic the transition state of the sugar donor in the enzymatic reaction catalysed by chitin synthetase, is described. Compound (I) was found to competitively inhibit chitin synthetase from Saccharomyces cerevisiae with respect to UDP-GlcNAc, but displayed minimal antifungal activity.     

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

Integration of the cytogenetic and genetic linkage maps of Brassica oleracea

We have assigned all nine linkage groups of a Brassica oleracea genetic map to each of the nine chromosomes of the karyotype derived from mitotic metaphase spreads of the B. oleracea var. alboglabra line A12DHd using FISH. The majority of probes were BACs, with A12DHd DNA inserts, which give clear, reliable FISH signals. We have added nine markers to the existing integrated linkage map, distributed over six linkage groups. BACs were definitively assigned to linkage map positions through development of locus-specific PCR assays. Integration of the cytogenetic and genetic linkage maps was achieved with 22 probes representing 19 loci. Four chromosomes (2, 4, 7, and 9) are in the same orientation as their respective linkage groups (O4, O7, O8, and O6) whereas four chromosomes (1, 3, 5, and 8) and linkage groups (O3, O9, O2, and O1) are in the opposite orientation. The remaining chromosome (6) is probably in the opposite orientation. The cytogenetic map is an important resource for locating probes with unknown genetic map positions and is also being used to analyze the relationships between genetic and cytogenetic maps.     

Cloning advances and challenges for conservation

Although controversy surrounds cloning efforts, the cloning of animals to assist efforts to preserve genetic variation in support of endangered species conservation efforts has attracted serious interest. A recent report by Loi et al. describing the cloning of a mouflon (a species of wild sheep) in a domestic sheep surrogate points to potential conservation opportunities and additional challenges in the evaluation of appropriate technologies for present and future efforts to conserve gene pools of endangered species.