Quantitative fine-tuning of photoreceptor cis-regulatory elements through affinity modulation of transcription factor binding sites.

Given the remarkable recent progress in gene therapy-based treatments for retinal disease, there is an urgent need for the development of new approaches to quantitative design and analysis of photoreceptor-specific promoters. In this study, we determined the relative binding affinity of all single-nucleotide variants of the consensus binding site of the mammalian photoreceptor transcription factor, Crx. We then showed that it is possible to use these data to accurately predict the relative binding affinity of Crx for all possible 8 bp sequences. By rationally adjusting the binding affinity of three Crx sites, we were able to fine-tune the expression of the rod-specific Rhodopsin promoter over a 225-fold range in living retinas. In addition, we showed that it is possible to fine-tune the activity of the rod-specific Gnat1 promoter over ∼275-fold range by modulating the affinity of a single Crx-binding site. We found that the action of individual binding sites depends on the precise promoter context of the site and that increasing binding affinity does not always equate with increased promoter output. Despite these caveats, this tuning approach permits quantitative engineering of photoreceptor-specific cis-regulatory elements, which can be used as drivers in gene therapy vectors for the treatment of blindness.

Randomized trial of IV dexamethasone for acute migraine in the emergency department.

BACKGROUND: It is not yet clear if corticosteroids are useful for the treatment of migraine. We determined the efficacy of 10 mg of IV dexamethasone as adjuvant therapy for patients presenting to an emergency department (ED) with acute migraine. METHODS: This was a randomized, double-blind, placebo-controlled multicenter trial. Subjects were randomized to dexamethasone 10 mg IV or placebo. As primary treatment for their migraine, all subjects received IV metoclopramide. Our primary hypotheses were the following: a greater percentage of patients with migraine who received dexamethasone would 1) achieve a headache-free state in the ED and maintain it for 24 hours and 2) have no headache-related functional impairment after ED discharge when compared to placebo. RESULTS: A total of 656 patients were approached for participation and 205 were randomized. The persistent pain-free outcome was achieved in 25% of those randomized to dexamethasone and 19% of placebo (p = 0.34). No functional impairment after ED discharge occurred in 67% of those randomized to dexamethasone and 59% of placebo (p = 0.20). In the subgroup of subjects with migraine lasting longer than 72 hours, 38% of those randomized to dexamethasone were persistently pain-free vs 13% of placebo (p = 0.06). Side effect profiles were similar, with the exception of acute medication reactions, which occurred more commonly in the dexamethasone group. CONCLUSION: A moderate dose of IV dexamethasone should not be administered routinely for the emergency department-based treatment of acute migraine, although it might be useful for patients with migraine lasting longer than 72 hours.

A trial of metoclopramide vs sumatriptan for the emergency department treatment of migraines.

OBJECTIVE: To compare the efficacy of 20 mg of IV metoclopramide, given up to four times over 2 hours as needed for persistent headache, with 6 mg of subcutaneous sumatriptan for the emergency department treatment of migraine headaches. METHODS: This was a randomized, double-blind, clinical trial with two intervention arms. The primary endpoint was change in pain intensity as measured by an 11-point pain scale at 2 hours. Secondary endpoints included change in pain intensity at 24 hours and rates of pain-free headache relief at 2 and 24 hours. RESULTS: Two hundred two patients were screened, and 78 of 91 eligible patients were randomized. The two groups had comparable pain scores at baseline. By 2 hours, the change in pain intensity for the metoclopramide group was 7.2 compared with 6.3 for the sumatriptan group (95% CI for difference: -0.2 to 2.2). When compared at 24 hours, the metoclopramide group had improved by 6.1 compared with baseline and the sumatriptan group had improved by 5.0 (95% CI for difference: -0.6 to 2.8). At 2 hours, pain-free rates were 59% in the metoclopramide arm and 35% in the sumatriptan arm (95% CI for difference of 24%: 2 to 46%). The most common side effects at both time points were weakness, dizziness, and drowsiness, which were distributed evenly between the two groups. There were no reports of chest pain within the first 2 hours. The incidence of restlessness, stiffness, and abnormal movements was distributed equally between the two groups. CONCLUSIONS: When compared at 2 and 24 hours, aggressive (20 mg dosed up to four times) IV metoclopramide and 6 mg of subcutaneous sumatriptan relieved migraine headache pain comparably. Some secondary endpoints suggest that metoclopramide may be the preferable therapy for migraines presenting to the emergency department.

Limited usefulness of initial blood cultures in community acquired pneumonia.

OBJECTIVE: The incidence of community acquired pneumonia (CAP) is about 4 million cases per year, with a hospitalisation rate of 20%. In non-immunocompromised patients hospitalised for CAP the rate of bacteraemia is less than 7% with predictable pathogens. Despite this, guidelines still recommend use of blood cultures (BCs) to direct treatment. This study tested the primary hypothesis that the proportion of false positive BCs would exceed the proportion of true positives. A secondary aim was to quantify the frequency with which antibiotic therapy was changed based on BC results. METHOD: Consecutive adults hospitalised from an urban emergency department (ED) with CAP between January 1999 and March 2001 were assessed retrospectively for study eligibility. Those with an infiltrate consistent with pneumonia on the admission chest radiograph and at least one set of BCs taken in the ED before antibiotics were given were entered into the study. Patients hospitalised within the previous two weeks, nursing home residents, and immunosuppressed patients were excluded. RESULTS: 821 patients were admitted for CAP and 355 met inclusion criteria. The proportion of false positive BCs (10%) exceeded the proportion of true positives (9%), by 1% (95%CI -3.3% to 5.5%). Antibiotic therapy was changed on the basis of BC results in 5% of patients (95%CI 3% to 8%). CONCLUSION: The rate of false positive BCs in patients hospitalised with CAP is similar to the rate of true positives. BCs only infrequently lead to changes in antibiotic therapy, and in no instance were therapeutic changes driven by detection of resistant organisms. The results question the utility of routine BCs in immunocompetent patients with CAP.

Energy dependence of particle multiplicities in central Au+Au collisions.

We present the first measurement of the pseudorapidity density of primary charged particles in Au+Au collisions at root square[s(NN)] = 200 GeV. For the 6% most central collisions, we obtain dN(ch)/d(eta)/(/eta/<1) = 650+/-35(syst). Compared to collisions at root square[s(NN)] = 130 GeV, the highest energy studied previously, an increase by a factor of 1.14+/-0.05 at 90% confidence level, is found. The energy dependence of the pseudorapidity density is discussed in comparison with data from proton-induced collisions and theoretical predictions.

Randomized clinical trial of intravenous magnesium sulfate as an adjunctive medication for emergency department treatment of migraine headache.

STUDY OBJECTIVE: We test the hypothesis that intravenous magnesium sulfate is an effective adjunctive medication for treatment of acute migraine. METHODS: In this randomized, double-blind, placebo-controlled trial, adults presenting to 2 urban emergency departments with headache meeting International Headache Society criteria for acute migraine received either 20 mg of intravenous metoclopramide plus 2 g of intravenous magnesium sulfate or 20 mg of intravenous metoclopramide plus a placebo of intravenous saline solution at 15-minute intervals for a maximum of 3 doses or until pain relief occurred. At 0, 15, 30, and 45 minutes, patients recorded pain intensity using a standard visual analog scale (VAS). The primary study end point was the between-group difference in pain improvement when initial and final VAS scores were compared. RESULTS: Of 44 patients enrolled (21 randomized to metoclopramide plus magnesium and 23 to metoclopramide plus placebo), 42 (95%) were women. Baseline features were comparable in both groups. Each group experienced a more than 50-mm improvement in VAS score during the study. However, this improvement was smaller in the magnesium group for the primary end point (16-mm difference favoring placebo [95% confidence interval (CI) -2 to 34 mm]), as was the proportion with normal functional status at their final rating (36% absolute difference also favoring placebo [95% CI 7% to 65%]). Using a 50% reduction in pain to dichotomize VAS scores, the number needed to harm with magnesium plus metoclopramide versus metoclopramide alone is 4 patients (95% CI 2 to 36). CONCLUSION: Although this result was unexpected, our data suggest that the addition of magnesium to metoclopramide may attenuate the effectiveness of metoclopramide in relieving migraine. Countertherapeutic cerebral vasodilatation caused by magnesium is a plausible, although unproven, explanation for this finding. Because of the preponderance of women in our trial, these data may not be generalizable to men.

The regulation of forkhead/HNF-3beta expression in the Ciona embryo.

The Ciona forkhead/HNF-3beta gene (Ci-fkh) is expressed in the primary axial tissues of the developing tadpole, including the notochord, endoderm, and rudimentary floor plate of the CNS. In an effort to determine the basis for this complex pattern of expression we have conducted a detailed analysis of the Ci-fkh 5'-regulatory region. Different 5' sequences were attached to a lacZ reporter gene and analyzed in electroporated Ciona embryos. A short regulatory sequence (AS) located approximately 1.7 kb upstream of the transcribed region is shown to be essential for expression in all three axial tissues. The proximal 20 bp of the AS contains overlapping Snail repressor elements and a T-box motif. Deleting these sequences causes the loss of reporter gene expression in the endoderm, as well as expanded expression in the neural tube. These results suggest that a T-box gene such as Ci-VegTR activates Ci-fkh expression in the endoderm, while the Ci-Sna repressor excludes expression from the lateral ependymal cells and restricts the Ci-fkh pattern to the rudimentary floor plate in ventral regions of the neural tube. We also present evidence for Ci-fkh positive autofeedback, whereby the Ci-Fkh protein binds to critical activator sites within the Ci-fkh 5'-regulatory region and helps maintain high levels of expression. We discuss these results with respect to forkhead/HNF-3beta regulation in vertebrates.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

DCAMKL1 encodes a protein kinase with homology to doublecortin that regulates microtubule polymerization.

Doublecortin (DCX) is a microtubule-associated protein required for neuronal migration to the cerebral cortex. DCAMKL1 consists of an N terminus that is 65% similar to DCX throughout the entire length of DCX, but also contains an additional 360 amino acid C-terminal domain encoding a putative Ca(2+)/calmodulin-dependent protein kinase. The homology to DCX suggested that DCAMKL1 may regulate microtubules, as well as mediate a phosphorylation-dependent signal transduction pathway. Here we show that DCAMKL1 is expressed throughout the CNS and PNS in migrating neuronal populations and overlaps in its expression with DCX and microtubules. Purified DCAMKL1 associates with microtubules and stimulates polymerization of purified tubulin and the formation of aster-like microtubule structures. Overexpressed DCAMKL1 leads to striking microtubule bundling in cell lines and cultured primary neural cells. Time-lapse imaging of cells transfected with a DCAMKL1-green fluorescent protein fusion protein shows that the microtubules associated with the protein remain dynamic. DCAMKL1 also encodes a functional kinase capable of phosphorylating myelin basic protein and itself. However, elimination of the kinase activity of DCAMKL1 has no detectable effect on its microtubule polymerization activity. Because DCAMKL1 is coexpressed with DCX, the two proteins form a potentially mutually regulatory network linking calcium signaling and microtubule dynamics.

Suppressor of hairless activates brachyury expression in the Ciona embryo.

The Ciona Brachyury gene (Ci-Bra) is regulated, in part, by a 434-bp enhancer that mediates restricted expression in the notochord. Here we present evidence that a Ciona Suppressor of Hairless ¿Ci-Su(H)¿ protein functions as an activator of this enhancer. Point mutations that reduce the binding of a GST/Ci-Su(H) fusion protein in vitro diminish the expression of mutagenized Ci-Bra/lacZ transgenes in electroporated embryos. Overexpression of a Ci-Su(H) fusion protein containing the Drosophila Hairy repression domain interferes with notochord differentiation, producing mutant tadpoles with shortened tails. Expression of a constitutively activated Xotch receptor in the notochord, endoderm, and CNS also alters tail morphogenesis. These results suggest that a Notch-Su(H) pathway might participate in notochord differentiation in Ciona.

The snail repressor establishes a muscle/notochord boundary in the Ciona embryo.

Previous studies have identified a minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra), which is sufficient to direct a notochord-specific pattern of gene expression. Here we present evidence that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages.

Lineage-specific regulation of the Ciona snail gene in the embryonic mesoderm and neuroectoderm.

The snail gene encodes a highly conserved, zinc-finger transcription factor that has been implicated in the specification of mesodermal and neuronal tissues in a variety of organisms. In the ascidian, Ciona intestinalis, snail (Ci-sna) is expressed at the 32-cell stage in derivatives of the B4.1 blastomere, including B6.2, B6.4, and B7.5, which give rise to the primary-lineage tail muscles of the tadpole. At later stages, Ci-sna is expressed in the lineages that will form the secondary tail muscle, the lateral ependymal cells of the spinal cord, and the dorsal cells of the cerebral vesicle. A minimal, 504-bp cis-regulatory sequence from the Ci-sna promoter region, the B4.1 enhancer, is shown to direct the expression of heterologous promoters in primary-lineage muscles. Furthermore, evidence is presented that cis-regulatory elements necessary for expression in both the secondary muscle and neuronal lineages are separate from the B4.1 enhancer. We discuss the possibility that the classical muscle determinant present in ascidian eggs may correspond to bHLH activators, which bind to specific E-box sequences contained in the B4.1 enhancer.

Dorsoventral patterning of the vertebrate neural tube is conserved in a protochordate.

The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. Here we analyze the expression of HNF-3beta and snail homologues in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. A combination of in situ hybridization and promoter fusion analyses was used to demonstrate that the Ciona HNF-3beta homologue is expressed in the ventralmost ependymal cells of the neural tube, while the Ciona snail homologue is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. We propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians.

Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis.

We present evidence that the embryo of the ascidian, Ciona intestinalis, is an easily manipulated system for investigating the establishment of basic chordate tissues and organs. Ciona has a small genome, and simple, well-defined embyronic lineages. Here, we examine the regulatory mechanisms underlying the differentiation of the notochord. Particular efforts center on the regulation of a notochord-specific Ciona Brachyury gene (Ci-Bra). An electroporation method was devised for the efficient incorporation of transgenic DNA into Ciona embryos. This method permitted the identification of a minimal, 434 bp enhancer from the Ci-Bra promoter region that mediates the notochord-restricted expression of both GFP and lacZ reporter genes. This enhancer contains a negative control region that excludes Ci-Bra expression from inappropriate embryonic lineages, including the trunk mesenchyme and tail muscles. Evidence is presented that the enhancer is activated by a regulatory element which is closely related to the recognition sequence of the Suppressor of Hairless transcription factor, thereby raising the possibility that the Notch signaling pathway plays a role in notochord differentiation. We discuss the implications of this analysis with regard to the evolutionary conservation of integrative enhancers, and the subdivision of the axial and paraxial mesoderm in vertebrates.

Characterization of an immunodeficiency mutant in Drosophila.

Drosophila immunity and embryogenesis appear to be linked by an evolutionarily ancient signalling pathway, which includes the Rel-domain transcription factors Dif and dorsal, respectively, as well as a common inhibitor, cactus. Previous genetic screens have centered on maternal mutants that disrupt the dorsal pathway. In an effort to identify additional components that influence Rel-domain gene function we have conducted a search for immunodeficiency mutants in Drosophila. One such mutant, which maps near the Black cells (Bc) gene, causes a severe impairment of the normal immune response, including attenuated induction of several immunity genes. Survival assays indicate a positive correlation between the induction of these genes, particularly diptericin, and resistance to bacterial infection. These studies are consistent with the notion that insect anti-microbial peptides work synergistically by binding distinct targets within infecting pathogens. Evidence is also presented that non-specific acquired immunity results from the persistence of bacterial metabolites long after primary infection. We discuss the potential usefulness of this study with regard to the identification of conserved components of Rel signalling pathways.

beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria.

A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Cloning and characterization of a Rhizobium meliloti homolog of the Escherichia coli cell division gene ftsZ.

The ftsZ gene is essential for initiation of cell division in Escherichia coli and Bacillus subtilis. To begin our studies of division arrest during differentiation of Rhizobium meliloti bacteroids, we isolated a R. meliloti ftsZ homolog, ftsZRm. Degenerate primers directed towards a conserved region of ftsZ were used to amplify a segment of R. meliloti DNA by polymerase chain reaction, and the product of this reaction was then used to isolate positive clones from a bacteriophage library. The DNA sequence of an open reading frame containing the region of homology indicated that the R. meliloti FtsZ protein (FtsZRm) is 50% homologous to the known E. coli and B. subtilis FtsZ proteins, but at 590 amino acids (63 kDa), it is predicted to be nearly 50% larger. Strong expression of an approximately 70-kDa labeled protein in a coupled in vitro transcription-translation system supports this prediction. The additional 200 amino acids appear to fall in a single internal domain highly enriched for proline and glutamine residues. When we regulated R. meliloti ftsZ (ftsZRm) expression on a high-copy-number plasmid in E. coli with Plac and laclq, cells were smaller than normal in the presence of low FtsZRm levels (with no isopropyl-beta-D-thiogalactopyranoside [IPTG]) and filamentous when FtsZRm was overproduced (with IPTG). These results suggest that low levels of FtsZRm stimulate E. coli cell division, while high levels may be inhibitory.