Water Deficit and Spatial Pattern of Leaf Development. Variability in Responses Can Be Simulated Using a Simple Model of Leaf Development1

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695–703).

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

Memory and long-term potentiation (LTP) dissociated: Normal spatial memory despite CA1 LTP elimination with Kv1.4 antisense

Long-term potentiation (LTP) in the hippocampal slice preparation has been proposed as an in vitro model for long-term memory. However, correlation of LTP with memory in living animals has been difficult to demonstrate. Furthermore, in the last few years evidence has accumulated that dissociate the two. Because potassium channels might determine the weight of synapses in networks, we studied the role of Kv1.4, a presynaptic A-type voltage-dependent K+ channel, in both memory and LTP. Reverse transcription–PCR and Western blot analysis with specific antibodies showed that antisense oligodeoxyribonucleotide to Kv1.4 microinjected intraventricularly into rat brains obstructed hippocampal Kv1.4 mRNA, “knocking down” the protein in the hippocampus. This antisense knockdown had no effect on rat spatial maze learning, memory, or exploratory behavior, but eliminated both early- and late-phase LTP and reduced paired-pulse facilitation (a presynaptic effect) in CA1 pyramidal neurons without affecting dentate gyrus LTP. This presynaptic Kv1.4 knockdown together with previous postsynaptic Kv1.1 knockdown demonstrates that CA1 LTP is neither necessary nor sufficient for rat spatial memory.

A transgenic mouse model with an inducible skin blistering disease phenotype

One of the current limitations of gene transfer protocols involving mammalian genomes is the lack of spatial and temporal control over the desired gene manipulation. Starting from a human keratin gene showing a complex regulation as a template, we identified regulatory sequences that confer inducible gene expression in a subpopulation of keratinocytes in stratified epithelia of adult transgenic mice. We used this cassette to produce transgenic mice with an inducible skin blistering phenotype mimicking a form of epidermolytic hyperkeratosis, a keratin gene disorder. Upon induction by topical application of a phorbol ester, the mutant keratin transgene product accumulates in the differentiating layers of epidermis, leading to keratinocyte lysis after application of mechanical trauma. This mouse model will allow for a better understanding of the complex relationship between keratin mutation, keratinocyte cytoarchitecture, and hypersensitivity to trauma. The development of an inducible expression vector showing an exquisite cellular specificity has important implications for manipulating genes in a spatially and temporally controlled fashion in transgenic mice, and for the design of gene therapy strategies using skin as a tissue source for the controlled delivery of foreign substances.

A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.

To test whether the structure of a protein is determined in a manner akin to the assembly of a jigsaw puzzle, up to 10 adjacent residues within the core of T4 lysozyme were replaced by methionine. Such variants are active and fold cooperatively with progressively reduced stability. The structure of a seven-methionine variant has been shown, crystallographically, to be similar to wild type and to maintain a well ordered core. The interaction between the core residues is, therefore, not strictly comparable with the precise spatial complementarity of the pieces of a jigsaw puzzle. Rather, a certain amount of give and take in forming the core structure is permitted. A simplified hydrophobic core sequence, imposed without genetic selection or computer-based design, is sufficient to retain native properties in a globular protein.

Profluorescent protease substrates: intramolecular dimers described by the exciton model.

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.

Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.