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Identification of new central nervous system specific mouse microRNAs.

MicroRNAs (miRNAs) are small regulatory molecules suppressing mRNA activity in metazoans. Here we describe two new miRNAs cloned from brain tissue of mouse embryos. These miRNAs are expressed mainly during embryogenesis and specifically in the central nervous system. We also established the expression patterns of three recently identified miRNAs that were found in our short RNA library. All of them were expressed in the brain and spinal chord but while miR-410 and miR-431 were central nervous system specific, miR-500 was also expressed in limb buds. In addition, the expression of miR-500 in limb buds showed very strong asymmetry in favour of the left hand side.

Animals↗

HIP/PAP gene, encoding a C-type lectin overexpressed in primary liver cancer, is expressed in nervous system as well as in intestine and pancreas of the postimplantation mouse embryo.

We originally isolated the HIP/PAP gene in a differential screen of a human hepatocellular carcinoma cDNA library. This gene is expressed at high levels in 25% of primary liver cancers but not in nontumorous liver. HIP/PAP belongs to the family of C-type lectins and acts as an adhesion molecule for hepatocytes. In normal adult human tissues, HIP/PAP expression is found in pancreas (exocrine and endocrine cells) and small intestine (Paneth and neuroendocrine cells). In order to gain insight into the possible role of HIP/PAP in vivo, we have investigated the pattern of HIP/PAP expression in the developing postimplantation mouse embryo by in situ hybridization. Detailed analysis of developing mouse embryos revealed that HIP/PAP gene exhibits a restricted expression pattern during development. Thus, HIP/PAP transcripts are first observed within the nervous system from day 14.5 onwards in trigeminal ganglia, dorsal root ganglia, and spinal cord where it appears to be an early specific marker of a subpopulation of motor neurons. At laster stages, HIP/PAP transcripts were detected in intestine and pancreas at day 16.5 but not in embryonic liver. This highly restricted expression pattern suggests that HIP/PAP might participate in neuronal as well as intestinal and pancreatic cell development.

Animals↗

Small molecule inhibitors of bacterial quorum sensing and biofilm formation.

Bacteria monitor their local population densities using small molecules (or autoinducers) in a process known as quorum sensing. Here, we report a new and efficient synthetic route to naturally occurring bacterial autoinducers [N-acyl l-homoserine lactones (AHLs)] that is readily amenable to the synthesis of analogues. This route has been applied in the first synthesis of a library of non-native AHLs. Evaluation of these compounds in bacterial reporter gene and biofilm assays has revealed a potent set of quorum sensing antagonists. These ligands will serve as valuable new tools to explore the role of quorum sensing in bacterial pathogenesis.

Bacterial Proteins↗

Combinatorial methods for small-molecule placement in computational enzyme design.

The incorporation of small-molecule transition state structures into protein design calculations poses special challenges because of the need to represent the added translational, rotational, and conformational freedoms within an already difficult optimization problem. Successful approaches to computational enzyme design have focused on catalytic side-chain contacts to guide placement of small molecules in active sites. We describe a process for modeling small molecules in enzyme design calculations that extends previously described methods, allowing favorable small-molecule positions and conformations to be explored simultaneously with sequence optimization. Because all current computational enzyme design methods rely heavily on sampling of possible active site geometries from discrete conformational states, we tested the effects of discretization parameters on calculation results. Rotational and translational step sizes as well as side-chain library types were varied in a series of computational tests designed to identify native-like binding contacts in three natural systems. We find that conformational parameters, especially the type of rotamer library used, significantly affect the ability of design calculations to recover native binding-site geometries. We describe the construction and use of a crystallographic conformer library and find that it more reliably captures active-site geometries than traditional rotamer libraries in the systems tested.

Catalytic Domain↗

Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene.

A full-length placental alkaline phosphatase (PLAP) cDNA was used to identify and clone the PLAP-like Nagao isozyme gene from human genomic libraries. The entire nucleotide sequence of the gene reveals the existence of 11 exons interrupted by 10 small introns (76-427 base pairs). Putative regulatory sequences have been identified in the promoter regions as well as dispersed in the introns. The deduced amino acid sequence of the Nagao isozyme indicates that the mature molecule is composed of 513 amino acids, of which 12 residues are different from the sequence of PLAP (98% homology). A sequence derived from exon III of the Nagao isozyme gene was used to synthesize a peptide (NH2-Lys-Leu-Gly-Pro-Glu-Thr-Phe-Leu-Ala-COOH) that contains two mutations with respect to the corresponding PLAP sequence. This peptide elicited rabbit polyclonal antibodies that reacted specifically with the seminoma Nagao isozyme but not with PLAP in electrophoretic transfer blots. These results indicate that the tumor, and possibly the normal testis, Nagao isozyme is encoded by a gene referred to as germ-cell alkaline phosphatase gene that differs from the PLAP gene expressed by syncitiotrophoblastic cells.

Alkaline Phosphatase↗

From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops.

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?

Base Sequence↗

DNA and RNA aptamers as modulators of protein function.

The SELEX technique (systematic evolution of ligands by exponential enrichment) is a combinatorial library approach in which DNA or RNA molecules are selected by their ability to bind their protein targets with high affinity and specificity. The isolated molecules are referred to as aptamers (from aptus = Latin "to fit"). First, RNA and DNA aptamers were identified that bind to proteins naturally interacting with nucleic acids, or to small organic molecules such as ATP. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands for a wide range of proteins of importance for therapy, and diagnostics. Since these RNA and DNA molecules bind their targets with similar affinities as antibodies, and are able to distinguish between isotypes of an enzyme, aptamers have been also called synthetic antibodies. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as receptors that are only functional in their membrane-bound form, cells, and trypanosomes. RNA aptamers have been expressed in living cells where they inhibit a protein implicated in intracellular signal transduction. The utility of aptamers for in vivo experiments, and diagnostic and therapeutic purposes, is considerably enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Recently, such inhibitors have been evolved for a great variety of targets, including receptors, growth factors, and adhesion molecules implicated in disease. Furthermore, some results were already obtained in animal models and clinical trials.

Animals↗

Molecular cloning of higher-plant 3-oxoacyl-(acyl carrier protein) reductase. Sequence identities with the nodG-gene product of the nitrogen-fixing soil bacterium Rhizobium meliloti.

cDNA clones encoding the fatty-acid- biosynthetic enzyme NADPH-linked 3-oxoacyl-(acyl carrier protein) (ACP) reductase were isolated from a Brassica napus (rape) developing seed library and from an Arabidopsis thaliana (thale cress) leaf library. The N-terminal end of the coding region shows features typical of a stromal-targeting plastid-transit peptide. The deduced amino acid sequences have 41% and 55% identity respectively with the nodG-gene product of Rhizobium meliloti, one of the host-specific genes that restrict infectivity of this bacterium to a small range of host plants. The probability that the nodG-gene product is a oxoreductase strengthens the hypothesis that some of the host-specific nod-gene products are enzymes which synthesize polyketides that uniquely modify the Rhizobium nodulation signal molecule.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

Synthesis of ligand-specific phage-display ScFv against the herbicide picloram by direct cloning from hyperimmunized mouse.

Immunoglobulin genes were directly isolated from the splenocytes of a BALB/C mouse hyperimmunized with the auxinic herbicide picloram conjugated to bovine serum albumin. Variable light and heavy domain DNA were joined to produce single-chain Fv (scFv) DNA, which was cloned into phage vector fd-tet-GIIID to display multiple copies of scFv on the filamentous phage minor coat protein gIIIp. The phage-display scFv library (10(4) clones) was selected against picloram conjugated to ovalbumin. After five rounds of panning, individual clones were analyzed. ScFv with different affinities to picloram (IC(50) values ranging from 20 ppb to 10 ppm) were detected in the final enriched pool. The increased avidity of the phage vector enhanced the selection (i.e., panning) of multiple picloram-specific recombinant antibodies. Stringent selection was required to isolate the clones with the highest affinity. Nucleotide sequence analysis of six isolated clones revealed that all of the V(L) belonged to the V kappa 9A family joined to J kappa 2 segments. All of the V(H) belonged to the V(H)()7183 family and joined to two different J segments (i.e., J(H)()2 or J(H)()4). Different from the immune response to large molecular weight molecules (MW > 10,000 Da), which requires both VDJ segment rearrangement and somatic hypermutations, production of high-affinity antibodies to picloram, a small ligand having a formula weight of 241.5 Da, predominantly requires somatic hypermutations.

Animals↗

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.

Animals↗

Chemical microarray: a new tool for drug screening and discovery.

HTS with microtiter plates has been the major tool used in the pharmaceutical industry to explore chemical diversity space and to identify active compounds and pharmacophores for specific biological targets. However, HTS faces a daunting challenge regarding the fast-growing numbers of drug targets arising from genomic and proteomic research, and large chemical libraries generated from high-throughput synthesis. There is an urgent need to find new ways to profile the activity of large numbers of chemicals against hundreds of biological targets in a fast, low-cost fashion. Chemical microarray can rise to this challenge because it has the capability of identifying and evaluating small molecules as potential therapeutic reagents. During the past few years, chemical microarray technology, with different surface chemistries and activation strategies, has generated many successes in the evaluation of chemical-protein interactions, enzyme activity inhibition, target identification, signal pathway elucidation and cell-based functional analysis. The success of chemical microarray technology will provide unprecedented possibilities and capabilities for parallel functional analysis of tremendous amounts of chemical compounds.

Animals↗

Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein.

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

Allosteric Regulation↗

Therapeutic peptides and peptidomimetics.

Peptidomimetics are one set of probes used in the transition pathway of small molecule drug design. Cyclization of the peptide backbone and its modification with aromatic residues constitutes an effective approach to mimetic drug design and circumvents obstacles associated with delivery and formulation of peptides and peptidomimetics. In the past year, examples of mimicking beta-turn structures has led to combining design strategies with molecular libraries, demonstrating that peptidomimetics can provide valuable clues about receptor similarities not revealed by their endogenous ligands. This information can lead to the development of dual inhibitors. In addition, this work suggests that the use of libraries and rational design need not be mutually exclusive approaches to lead discovery.

Animals↗

Sulfotransferase structural biology and inhibitor discovery.

Sulfotransferases catalyze the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to proteins, carbohydrates and small molecules. The sulfotransferases comprise cytosolic and Golgi-resident enzymes; Golgi-resident enzymes represent fertile territory for identifying pharmaceutical targets. Structure-based sequence alignments indicate that the structural fold, and the PAPS-binding site, is conserved between the two classes. Initial efforts to identify sulfotransferase inhibitors by screening kinase inhibitor libraries yielded competitive inhibitors of PAPS with muM IC(50) values. Within particular classes of Golgi-resident sulfotransferases that show tight in vitro specificity, the substrate-binding site might be a suitable drug target, although sulfotransferases are generally assumed to be difficult to inhibit as a result of the expected size and chemical character of the substrate-binding site.

Adenosine Diphosphate↗

Development of a cyanovirin-N-HIV-1 gp120 binding assay for high throughput screening of natural product extracts by time-resolved fluorescence.

The unique, high-affinity binding of cyanovirin-N (CV-N), a potent anti-human immunodeficiency virus (HIV) protein, to the HIV envelope glycoprotein gp120, was exploited to develop an HTS assay in an attempt to discover small-molecule mimetics of CV-N. A competition binding assay was developed using CV-N labeled with europium (Eu(3+)). The labeling protocol did not significantly alter the gp120 binding properties or the antiviral activity of CV-N. This report describes the assay development, validation, and results of screening a large library of aqueous and organic natural product extracts. The extracts were incubated with immobilized recombinant gp120 in 96-well plates prior to the addition of Eu(3+)-labeled CV-N. Following a wash step, bound CV-N was measured by dissociation-enhanced time-resolved fluorometry of Eu(3+). The assay proved to be robust, rapid, and reproducible, and was used to screen over 50,000 natural product extracts, and has resulted in the identification of several aqueous natural product extracts that inhibited CV-N-gp120 binding and also had anti-HIV activity.

Anti-HIV Agents↗

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon.

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.

Amino Acid Sequence↗

Structure, sequence and expression of the mouse Cx43 gene encoding connexin 43.

Gap junctions, membrane channels that mediate the diffusion of ions and small molecules between cells, are hypothesized to play a role in development and growth regulation. The Cx43 gene (encoding connexin 43) is one member of the gap junction gene family whose transcripts are expressed in a highly regionalized manner during mouse development. We cloned and sequenced Cx43 cDNAs from a 7.5-day mouse embryo cDNA library. These cDNA clones encode the authentic 43-kDa connexin. Analysis of RNA isolated from different regions of the 7.5-day mouse embryo revealed that Cx43 transcripts are differentially expressed, with expression detected in the embryo proper, but not in the extraembryonic region containing the ectoplacental cone. Using one of the newly isolated mouse Cx43 cDNA probes, we screened a mouse genomic DNA library and cloned the Cx43 gene. Restriction mapping and sequencing of the cloned genomic inserts revealed that Cx43 contains two exons and a 10.5-kb intron located in the 5' untranslated region (5'-UTR). We mapped the Cx43 transcription start point (tsp) by RNase protection and primer extension analyses and showed that transcripts expressed in the 7.5-day mouse embryo and in adult tissues are initiated from the same tsp. The DNA sequence immediately upstream from the tsp contains a putative AP1-binding site and a degenerate TATA consensus sequence. A comparison of mouse, rat, human and bovine Cx43s showed that the 3'-UTR has an unexpectedly high degree of sequence homology. This includes conservation of four AUUUA motifs, a sequence associated with transcript instability in immediate early genes.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

Genetic loci of Streptococcus mitis that mediate binding to human platelets.

The direct binding of bacteria to platelets is a postulated major interaction in the pathogenesis of infective endocarditis. To identify bacterial components that mediate platelet binding by Streptococcus mitis, we screened a Tn916deltaE-derived mutant library of S. mitis strain SF100 for reduced binding to human platelets in vitro. Two distinct loci were found to affect platelet binding. The first contains a gene (pblT) encoding a highly hydrophobic, 43-kDa protein with 12 potential membrane-spanning segments. This protein resembles members of the major facilitator superfamily of small-molecule transporters. The second platelet binding locus consists of an apparent polycistronic operon. This region includes genes that are highly similar to those of Lactococcus lactis phage r1t and Streptococcus thermophilus phage 01205. Two genes (pblA and pblB) encoding large surface proteins are also present. The former encodes a 107-kDa protein containing tryptophan-rich repeats, which may serve to anchor the protein within the cell wall. The latter encodes a 121-kDa protein most similar to a tail fiber protein from phage 01205. Functional mapping by insertion-duplication mutagenesis and gene complementation indicates that PblB may be a platelet adhesin and that expression of PblB may be linked to that of PblA. The combined data indicate that at least two genomic regions contribute to platelet binding by S. mitis. One encodes a probable transmembrane transporter, while the second encodes two large surface proteins resembling structural components of lysogenic phages.

Amino Acid Sequence↗