Optimal control approach to gradient-index design for beam reshaping.

We address the problem of reshaping light in the Schrödinger optics regime from the perspective of the optimal control theory. In technological applications, Schrödinger optics is often used to model a slowly varying amplitude of a para-axially propagating electric field where the square of the waveguide's index of refraction is treated as the potential. The objective of the optimal control problem is to find the controlling potential which, together with the constraining Schrödinger dynamics, optimally reshapes the intensity distribution of Schrödinger eigenfunctions from one end of the waveguide to the other. This work considers reshaping problems found in work by Kunkel and Leger, and addresses computational needs by adopting tools from the quantum control literature. The success of the optimal control approach is demonstrated numerically.

Reduction-based strategy for optimal control of Bose-Einstein condensates.

Applications of Bose-Einstein condensates (BEC) often require that the condensate be prepared in a specific complex state. Optimal control is a reliable framework to prepare such a state while avoiding undesirable excitations, and, when applied to the time-dependent Gross-Pitaevskii equation (GPE) model of BEC in multiple space dimensions, results in a large computational problem. We propose a control method based on first reducing the problem, using a Galerkin expansion, from a partial differential equation to a low-dimensional Hamiltonian ordinary differential equation system. We then apply a two-stage hybrid control strategy. At the first stage, we approximate the control using a second Galerkin-like method known as the chopped random basis to derive a finite-dimensional nonlinear programing problem, which we solve with a differential evolution algorithm. This search method then yields a candidate local minimum which we further refine using a variant of gradient descent. This hybrid strategy allows us to greatly reduce excitations both in the reduced model and the full GPE system.

Thyrotropin-releasing hormone precursor: characterization in rat brain.

To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.

Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice.

Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.

Calcitonin messenger RNA encodes multiple polypeptides in a single precursor.

Recombinant DNA techniques were used to analyze the structure of the messenger RNA encoding a precursor of calcitonin, a small calcium-regulating hormone of 32 amino acids. Analyses of the nucleotide sequences of cloned complementary DNA's comprising the entire coding sequence of the messenger RNA revealed that calcitonin is flanked at both its amino and carboxyl termini by peptide extensions linked to the hormone by short sequences of basic amino acids. The location of glycine next to the carboxyl terminal prolinamide of calcitonin is consistent with indications that glycine is required for the enzymatic amidation of proline to the prolinamide. During cellular biosynthesis, calcitonin arises from a large precursor protein by cleavages at both amino and carboxyl terminal residues of the hormone. These findings raise questions concerning the regulation of these cleavages and the potential biological functions of the precursor extensions derived from these cleavages.

Neuron-specific expression of the rat brain type II sodium channel gene is directed by upstream regulatory elements.

Genetic elements involved in cell-specific expression of the type II sodium channel gene were revealed using transient expression assays. A chimeric reporter gene containing 1051 bp of the sodium channel 5' flanking region was active in neuroblastoma and PC12 cells, but inactive in nonneuronal cell types. Deletion of upstream sequences resulted in an 80-fold increase in reporter gene activity in skeletal muscle cells, suggesting the presence of negative elements. Although no homologies were found between sequences in the type II 5' flanking region and other negative elements or "silencers," a small region common to the type II gene and other genes expressed in the nervous system was identified and may be involved in transcriptional regulation of neuronal genes.

Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel.

This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.

Acetylation of nuclear hormone receptor-interacting protein RIP140 regulates binding of the transcriptional corepressor CtBP.

CtBP (carboxyl-terminal binding protein) participates in regulating cellular development and differentiation by associating with a diverse array of transcriptional repressors. Most of these interactions occur through a consensus CtBP-binding motif, PXDLS, in the repressor proteins. We previously showed that the CtBP-binding motif in E1A is flanked by a Lys residue and suggested that acetylation of this residue by the p300/CBP-associated factor P/CAF disrupts the CtBP interaction. In this study, we show that the interaction between CtBP and the nuclear hormone receptor corepressor RIP140 is regulated similarly, in this case by p300/CBP itself. CtBP was shown to interact with RIP140 in vitro and in vivo through a sequence, PIDLSCK, in the amino-terminal third of the RIP140 protein. Acetylation of the Lys residue in this motif, demonstrated in vivo by using an acetylated RIP140-specific antibody, dramatically reduced CtBP binding. Mutation of the Lys residue to Gln resulted in a decrease in CtBP binding in vivo and a loss of transcriptional repression. We suggest that p300/CBP-mediated acetylation disrupts the RIP140-CtBP complex and derepresses nuclear hormone receptor-regulated genes. Disruption of repressor-CtBP interactions by acetylation may be a general mode of gene activation.

Histone binding protein RbAp48 interacts with a complex of CREB binding protein and phosphorylated CREB.

A CREB-CREB binding protein (CBP) complex was used as bait to screen a mouse embryo cDNA library in yeast. One of the strongest interactions identified the histone binding protein RbAp48. RbAp48 also interacted weakly with CBP alone but did not interact with phosphorylated or nonphosphorylated CREB. CBP (or its homologue p300) from HeLa cell nuclear extracts coimmunoprecipitated with RbAp48 and its homologue RbAp46 and bound to a glutathione S-transferase-RbAp48 fusion protein. This interaction was stimulated by the addition of phosphorylated CREB and allowed the association of core histones and mononucleosomes in an acetylation-dependent manner. RbAp48 lowered the K(m) of CBP histone acetylase activity and facilitated p300-mediated in vitro transcription of a chromatinized template in the presence of acetylcoenzyme A. These data indicate that the association of phosphorylated CREB with CBP promotes the binding of RbAp48 and its homologue RbAp46, allowing the formation of a complex that facilitates histone acetylation during transcriptional activation.

A cyclic AMP-responsive element-binding transcriptional activator in Drosophila melanogaster, dCREB-A, is a member of the leucine zipper family.

In this report, we describe the isolation and initial characterization of a Drosophila protein, dCREB-A, that can bind the somatostatin cyclic AMP (cAMP)-responsive element and is capable of activating transcription in cell culture. Sequence analysis demonstrates that this protein is a member of the leucine zipper family of transcription factors. dCREB-A is unusual in that it contains six hydrophobic residue iterations in the zipper domain rather than the four or five commonly found in this group of proteins. The DNA-binding domain is more closely related to mammalian CREB than to the AP-1 factors in both sequence homology and specificity of cAMP-responsive element binding. In embryos, dCREB-A is expressed in the developing salivary gland. A more complex pattern of expression is detected in the adult; transcripts are found in the brain and optic lobe cell bodies, salivary gland, and midgut epithelial cells of the cardia. In females, dCREB-A is expressed in the ovarian columnar follicle cells, and in males, dCREB-A RNA is seen in the seminal vesicle, ejaculatory duct, and ejaculatory bulb. These results suggest that the dCREB-A transcription factor may be involved in fertility and neurological functions.

Functional interaction between the coactivator Drosophila CREB-binding protein and ASH1, a member of the trithorax group of chromatin modifiers.

CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, we have begun a genetic analysis of CBP in flies. We have overproduced Drosophila melanogaster CBP (dCBP) in a variety of cell types and obtained distinct adult phenotypes. We used an uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, we showed that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. Our results thus implicate a second class of chromatin-associated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure.

Cubitus interruptus requires Drosophila CREB-binding protein to activate wingless expression in the Drosophila embryo.

CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophila homologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophila development, including the hedgehog (hh), decapentaplegic (dpp), and Toll pathways. Although dCBP is required for the expression of the hh target genes, wingless (wg) and patched (ptc) in vivo, and potentiates ci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by ci binding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary for ci-mediated transactivation of wg during Drosophila embryogenesis.

MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein.

A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB-CREB-binding protein (CBP) complex. When tested for binding to CREB or CBP individually, this MLL fragment interacted directly with CBP, but not with CREB. In vitro binding experiments refined the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain). The transactivation activity of MLL was dependent on CBP, as either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction proved effective at inhibiting MLL-mediated transactivation. Single amino acid substitutions within the MLL activation domain revealed that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, were critical for the interaction of MLL with CBP. Using purified components, we found that the MLL activation domain facilitated the binding of CBP to phosphorylated CREB. In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP.

Distinguishable promoter elements are involved in transcriptional activation by E1a and cyclic AMP.

The sequence motif CGTCA is critical for binding of a group of cellular transcription factors (ATF, CREB, E4F, and EivF) and for activation of certain E1a-inducible and cyclic AMP (cAMP)-inducible promoters. We have tested different promoter elements containing the CGTCA motif (referred to here as ATF-binding sites) for the ability to function as E1a or cAMP response elements. The adenovirus E4 promoter and the cellular vasoactive intestinal peptide (VIP) promoter responded differently to E1a and cAMP, demonstrating that the activating potential of ATF-binding sites within these promoters is not equivalent. While particular ATF-binding sites were sufficient for the activity of both the E4 (E1a inducibility) and VIP (cAMP inducibility) enhancers, these two enhancers had contrasting effects on E1a- and cAMP-inducible transcription. Thus, the relationship between E1a- and cAMP-inducible transcription is not simply explained by the action of these two inducers through the same promoter elements.

Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation.

Phosphorylation of the transcription factor CREB leads to the recruitment of the coactivator, CREB binding protein (CBP). Recent studies have suggested that CBP recruitment is not sufficient for CREB function, however. We have identified a conserved protein-protein interaction motif within the CBP-binding domains of CREB and another transcription factor, SREBP (sterol-responsive element binding protein). In contrast to CREB, SREBP interacts with CBP in the absence of phosphorylation. We have exploited the conservation of this interaction motif to test whether CBP recruitment to CREB is sufficient for transcriptional activation. Substitution of six nonconserved amino acids from SREBP into the activation domain of CREB confers high-affinity, phosphorylation-independent CBP binding. The mutated CREB molecule, CREB(DIEDML), activates transcription in F9 teratocarcinoma and PC12 cells even in the absence of protein kinase A (PKA). Addition of exogenous CBP augments the level of transcription mediated by CREB(DIEDML), and adenovirus 12S E1A blocks transcription, implicating CBP in the activation process. Thus, recruitment of CBP to CREB is sufficient for transcriptional activation. Addition of PKA stimulates transcription induced by CREB(DIEDML) further, suggesting that a phosphorylation event downstream from CBP recruitment augments CREB signaling.

Activation and repression in the nervous system.

The mechanisms underlying transcriptional activation and repression have become much clearer. Recent evidence suggests that transcription factors that do not bind DNA directly, the co-activators and co-repressors, mediate a large number of cell signaling events. Their association with histone acetylases, to mediate activation, or deacetylases, to mediate repression, provide a model for explaining how gene expression is regulated.

Glucagon precursors identified by immunoprecipitation of products of cell-free translation of messenger RNA.

Polyadenylated RNA extracted from anglerfish islets was translated in a wheat germ cell-free system containing [35S]methionine in the presence and absence of microsomal membranes prepared from a canine pancreas. Labeled translation products were analyzed by immunoprecipitation with an antiserum to porcine glucagon, followed by electrophoresis of the translation products and immunoprecipitated proteins on SDS polyacrylamide gels. In the absence of microsomal membranes two proteins of Mr = 14,500 and Mr = 12,500 were specifically immunoprecipitated with antiglucagon serum. Addition of microsomal membranes to the translation reactions resulted in a diminution of the labeled protein of Mr = 14,500 and a marked increase in the immunoreactive protein of Mr = 12,500. The protein of Mr = 12,500 was resistant to degradation by proteolytic enzymes added to translation reactions, indicating that it was segregated within microsomal vesicles. These results are consistent with synthesis of anglerfish islet glucagon in the form of a pre-prohormonal precursor (Mr = 14,500) containing a leader sequence that is cotranslationally cleaved from the protein by enzymes associated with microsomal membranes to produce a smaller intermediate prohormonal precursor (Mr = 12,500) of pancreatic glucagon (Mr = 3500).

The CRE-binding protein dCREB-A is required for Drosophila embryonic development.

We have previously described the cloning of a cyclic AMP response-element (CRE)-binding protein, dCREB-A, in Drosophila melanogaster that is similar to the mammalian CRE-binding protein CREB. dCREB-A is a member of the bZIP family of transcription factors, shows specific binding to the (CRE), and can activate transcription in cell culture. In this report, we describe the gene structure for dCREB-A, protein expression patterns throughout development and the necessary role for this gene in embryogenesis. The 4.5-kb transcript is encoded in six exons that are distributed over 21 kb of DNA. There are seven start sites and no TATA consensus sequences upstream. The dCREB-A protein is expressed in the nuclei of the embryonic salivary gland, proventriculus and stomadeum. Late in embryogenesis, tracheal cell nuclei and specific nuclei within the segments show staining with anti-dCREB-A antibodies. In adult female ovaries, dCREB-A is expressed in the stage 9 through stage 11 follicle cell nuclei. Null mutations of the dCREB-A gene give rise to animals that no longer express dCREB-A protein and die late in embryogenesis before or at hatching. The absolute requirement of dCREB-A for embryogenesis demonstrates a nonredundant function for a CRE-binding protein that will be useful in studying the role of specific signal transduction cascades in development.

CREB signaling--timing is everything.

Depolarization of neurons can lead to changes in gene expression that are important for such processes as synaptic plasticity, neuronal differentiation, and apoptosis. Impey and Goodman discuss some of the opposing models for how gene transcription in response to neuronal activity and elevations in intracellular calcium concentration is regulated. The pathways appear to converge on cyclic AMP response element-binding (CREB) protein, with the mitogen-activated protein kinase pathways playing an important role. The continuing debate about the involvement of calmodulin kinase IV is also described.

Fluorescence polarization analysis of protein-DNA and protein-protein interactions.

Fluorescence polarization is a powerful technique for characterizing macromolecular associations and can provide equilibrium determinations of protein-DNA and protein-protein interactions. This technique is particularly useful (and better suited than electrophoretic methods) to study low affinity protein-protein interactions. In this review, we have outlined the principles underlying the use of fluorescence polarization to study the assembly of higher order complexes that bind to the CRE. The availability of simple, relatively inexpensive instrumentation means that this technology is no longer only within the realm of the physical biochemist.