Adolescent neuroimmune function and its interaction with alcohol.

Adolescence is an evolutionarily conserved developmental period associated with behavioral change, including increased risk-taking and alcohol use. Experimentation with alcohol typically begins in adolescence and transitions to binge-like patterns of consumption. Alcohol exposure during adolescence can alter normative changes in brain structure and function. Understanding mechanisms by which ethanol impacts neurodevelopmental processes is important for preventing and ameliorating the deleterious consequences of adolescent alcohol abuse. This review focuses on the neuroimmune system as a key contributor to ethanol-induced changes in adolescent brain and behavior. After brief review of neuroimmune system development, acute and chronic effects of ethanol on adolescent neuroimmune functioning are addressed. Comparisons between stress/immunological challenges and ethanol on adolescent neuroimmunity are reviewed, as cross-sensitization is relevant during adolescence. The mechanisms by which ethanol alters neuroimmune functioning are then discussed, as they may portend development of neuropathological consequences and thus increase vulnerability to subsequent challenges and potentiate addictive behaviors.

Isolation and characterization of 2'-fluoro-, 2'-amino-, and 2'-fluoro-/amino-modified RNA ligands to human IFN-gamma that inhibit receptor binding.

CD4+ Th cells produce cytokines that play a pivotal role in the induction and regulation of cell-mediated and humoral immunity. Th1 cells, characterized by their secretion of IFN-gamma, induce macrophage cytotoxicity, delayed hypersensitivity, and enhanced cellular immunity. Secretion of IFN-gamma may even suppress Th2-enhanced humoral immunity. A counterproductive Th1 response and concomitant secretion of IFN-gamma may result in inflammatory and autoimmune diseases. IFN-gamma regulation of T cell function has potential for therapeutic intervention. To isolate high affinity oligonucleotide inhibitors of IFN-gamma activity, combinatorial libraries of RNA molecules modified at the 2' position of pyrimidine nucleotides with fluoro (F), amino (NH2), or a mixture of F and NH2 (2'-F/NH2) were screened using the SELEX (systematic evolution of ligands by exponential enrichment) combinatorial chemistry process. Each modified library of RNA molecules provides an expanded repertoire of molecules with increased structural diversity and unique binding properties. This added diversity increases the possibility of isolating molecules with the desired functional properties. These RNAs modified at the 2' position have also been shown to be nuclease resistant. High affinity ligands to human IFN-gamma from each modified library were isolated and characterized. The K(d)s of these ligands were determined and their secondary structures were predicted. The specificity of these ligands for IFN-gamma binding was confirmed, and their ability to inhibit binding of IFN-gamma to its receptor on A549 human lung carcinoma cells was determined. A 2'-NH2-modified ligand (2'-NH2-30) is described that binds IFN-gamma with high affinity and inhibits IFN-gamma-induced expression of MHC class I and ICAM-1 by human myeloid leukemia cells.

Potent 2'-amino-, and 2'-fluoro-2'-deoxyribonucleotide RNA inhibitors of keratinocyte growth factor.

Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing 2'amino-(2'NH2)- or 2'fluoro-(2'F)-2'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing 2'NH2 have lower melting temperatures (Tm) compared with helices containing 2'F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratinocyte growth factor from libraries containing modified RNA molecules with either 2'NH2 or 2'F pyrimidines. We demonstrated that 2'F RNA ligands have affinities (Kd approximately 0.3-3 pM) and bioactivities (Ki approximately 34 pM) superior to 2'NH2 ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, 2'F ligands have extreme thermo-stabilities (Tm approximately 78 degrees C in low salt, and specificities).

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Calcium-dependent oligonucleotide antagonists specific for L-selectin.

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.

Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations.

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants.

The control of plasmid ColE1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded RNAs. One RNA is the primer precursor and the other is a small counter-transcript called RNA I. The interaction of these highly structured RNAs results in inhibition of formation of mature primer RNA necessary for replication initiation. We have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibition by RNA I despite the fact that the mutations are located outside the overlap between primer and RNA I. We propose a model to account for the resistance of the mutant primers which is based on the differential folding of the nascent primer transcripts during transcription. We propose that the mutant primers diverge in structure from their wild-type counterparts during a discrete period during transcription. During this brief divergence, they are proposed to interact kinetically more slowly with RNA I than wild-type primer because a particular domain (the anti-tail) required for efficient interaction with RNA I is buried in a stem-loop structure while this same domain is predicted to be single-stranded in the wild-type. Despite substantial sequence divergence from ColE1, the primer precursors of the related plasmids CloDF13, RSF1030 and p15A also have retained the potential to expose their anti-tail in a similar manner to ColE1, suggesting that the folding pathway has been conserved in evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditional high copy number ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro.

We describe three independently isolated copy number mutants of a plasmid ColE1 derivative which undergo temperature- and growth-phase-dependent DNA amplification in Escherichia coli. These mutants have single base-pair alterations in a highly localized region of the plasmid genome encoding the replication primer RNA. The mutations map immediately upstream of the RNA1 transcript, altering the sequence between conserved elements of the RNA1 promoter. These mutants have 2- to 4-fold increased copy number relative to wild-type plasmids in exponential growth at 37 degrees C but undergo 20-fold amplification of copy number relative to wild-type when cells enter stationary phase. Cells containing these plasmids grow with normal kinetics at 37 degrees C but grow poorly at 42 degrees C. The poor growth is associated with high-level plasmid amplification. Both the temperature and growth phase plasmid DNA amplification are suppressed if the ColE1 rop gene product is provided in trans from a compatible plasmid. Analysis of steady-state RNA1 levels indicates that DNA amplification occurs in the presence of RNA1 made by the mutant plasmid. Thus, the DNA amplification of the copy mutant is not due to an inability to synthesize RNA1. Using an in vitro transcription system containing RNase H, we show that mutant primer processing by RNase H is resistant to levels of the replication initiation inhibitor RNA1 that inhibit wild-type primer processing. The defect in inhibition appears not to be at the level of association of RNA1 with nascent primer. These results indicate that mutant plasmid amplification is due to the ability of its primer precursor transcripts to serve as substrates for RNase H despite the presence of RNA1.

Transformation of Paramecium by microinjection of a cloned serotype gene.

Paramecia of a given serotype express only one of several possible surface proteins called immobilization antigens (i-antigens). A 16-kilobase plasmid containing the gene for immobilization antigen A from Paramecium tetraurelia, stock 51, was injected into the macronucleus of deletion mutant d12, which lacks that gene. Approximately 40% of the injected cells acquired the ability to express serotype A at 34 degrees C. Expression appeared to be regulated normally. The transformed cells, like wild type, could be switched to serotype B by antiserum treatment and culture at 19 degrees C; on transfer to 34 degrees C, they switched back to serotype A expression. Many of the lines retained the ability to express serotype A until autogamy, when the old macronucleus is replaced by a new one derived from the micronucleus. DNA from transformants contained the injected plasmid sequences, which were replicated within the paramecia. No evidence for integration was obtained. The majority of replicated plasmid DNA comigrated with a linearized form of the input plasmid. Nonetheless, the pattern of restriction fragments generated by transformant DNA and that generated by input plasmid DNA are identical and consistent with a circular rather than a linear map. These conflicting observations can be reconciled by assuming that a mixture of different linear fragments is present in the transformants, each derived from the circular plasmid by breakage at a different point. Copy-number determinations suggest the presence of 45,000-135,000 copies of the injected plasmid per transformed cell. These results suggest that the injected DNA contains information sufficient for both controlled expression and autonomous replication in Paramecium.

Construction of Co1E1 RNA1 mutants and analysis of their function in vivo.

We have carried out experiments designed to investigate the relationship between structure and function for the Co1E1 RNA1 species. RNA1 is a small RNA (108 nucleotides) that has been implicated in copy number control of the multicopy plasmid Co1E1. In vitro, RNA1 inhibits the processing of the primer precursor required for initiation of DNA replication. The RNA1 gene is entirely complementary to the 5'-terminal region of the primer. We have functionally separated these 2 RNA species by cloning the RNA1 gene downstream from the S. marcescens trp promoter. When cloned in a Co1E1-compatible plasmid, a trp-RNA1 fusion has been shown to mediate Co1E1-type incompatibility in vivo. The construction scheme described here also generates mutant RNA1 species with altered sequences at the 5' terminus of RNA1 which have been assayed for function in vivo. These experiments have indicated that sequences at the 5' terminus play a critical role in RNA1 function.

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo.

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens . The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5'-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3l nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5'-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5'-terminal region of RNA1 is crucial for its function.