Despite 30 years of effort, there is no effective vaccine for HIV-1. However, antibodies can prevent HIV-1 infection in humanized mice and macaques when passively transferred. New single-cell-based methods have uncovered many broad and potent donor-derived antibodies, and structural studies have revealed the molecular bases for their activities. The new data suggest why such antibodies are difficult to elicit and inform HIV-1 vaccine development efforts. In addition to protecting against infection, the newly identified antibodies can suppress active infections in mice and macaques, suggesting they could be valuable additions to anti-HIV-1 therapies and to strategies to eradicate HIV-1 infection.
The human IgG 2G12 recognizes high-mannose carbohydrates on the HIV-1
envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are
intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit
including a third antigen-binding interface not found in antibodies with
flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created
by intermolecular domain exchange, which exhibits increased breadth and
>50-fold increased neutralization potency compared with monomeric 2G12. The
four Fab and two Fc regions of dimeric 2G12 were localized at low resolution in
two independent structures, revealing IgG dimers with two (Fab)2 arms
analogous to the Fabs of conventional monomeric IgGs. Structures revealed three
conformationally-distinct dimers, demonstrating flexibility of the
(Fab)2–Fc connections that was confirmed by electron
microscopy, small-angle X-ray scattering, and binding studies. We conclude that
intermolecular domain exchange, flexibility, and bivalent binding to allow
avidity effects are responsible for the increased potency and breadth of dimeric
The cyclic nucleotides cAMP and cGMP are common signaling molecules synthesized in neurons following the activation of adenylyl or guanylyl cyclase. In the striatum, the synthesis and degradation of cAMP and cGMP is highly regulated as these second messengers have potent effects on the activity of striatonigral and striatopallidal neurons. This review will summarize the literature on cyclic nucleotide signaling in the striatum with a particular focus on the impact of cAMP and cGMP on the membrane excitability of striatal medium-sized spiny output neurons (MSNs). The effects of non-selective and selective phosphodiesterase (PDE) inhibitors on membrane activity and synaptic plasticity of MSNs will also be reviewed. Lastly, this review will discuss the implications of the effects PDE modulation on electrophysiological activity of striatal MSNs as it relates to the treatment of neurological disorders such as Huntington’s and Parkinson’s disease.
cyclic AMP; cyclic GMP; phosphodiesterase; electrophysiology
Broadly neutralizing antibodies (bNAbs) to HIV-1 envelope glycoprotein (Env) can
prevent infection in animal models. Characterized bNAb targets, although key to vaccine
and therapeutic strategies, are currently limited. We defined a new site of vulnerability
by solving structures of bNAb 8ANC195 complexed with monomeric gp120 by X-ray
crystallography and trimeric Env by electron microscopy. The site includes portions of
gp41 and N-linked glycans adjacent to the CD4 binding site on gp120,
making 8ANC195 the first donor-derived anti-HIV-1 bNAb with an epitope spanning both Env
subunits. Rather than penetrating the glycan shield using a single variable region CDR
loop, 8ANC195 inserted its entire heavy chain variable domain into a gap to form a large
interface with gp120 glycans and regions of the gp120 inner domain not contacted by other
bNAbs. By isolating additional 8ANC195 clonal variants, we identified a more potent
variant, which may be valuable for therapeutic approaches using bNAb combinations with
Mutating anti–HIV-1 broadly neutralizing antibodies increases their breadth and reduces pathways for viral escape through mutation.
Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46G54W, which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46G54W variants designed using analyses of the NIH45-46–gp120 complex structure and sequences of NIH45-46G54W–resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti–HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2–gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46G54W arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1YU-2–infected humanized mice, with viremic control exhibited when a third antibody, 10–1074, was added to the combination.
Eliminating key glycosylation sites on HIV envelope (Env) restores binding of the germline versions of known broadly neutralizing anti-Env antibodies.
Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti–CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
The existence of very potent, broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) offers the potential for prophylaxis against HIV-1 infection by passive immunization or gene therapy. Both routes permit the delivery of modified forms of IgGs. Smaller reagents are favored when considering ease of tissue penetration and the limited capacities of gene therapy vectors. Immunoadhesin (single-chain fragment variable [scFv]-Fc) forms of IgGs are one class of relatively small reagent that has been explored for delivery by adeno-associated virus. Here we investigated the neutralization potencies of immunoadhesins compared to those of their parent IgGs. For the antibodies VRC01, PG9, and PG16, the immunoadhesins showed modestly reduced potencies, likely reflecting reduced affinities compared to those of the parent IgG, and the VRC01 immunoadhesin formed dimers and multimers with reduced neutralization potencies. Although scFv forms of neutralizing antibodies may exhibit affinity reductions, they provide a means of building reagents with multiple activities. Attachment of the VRC01 scFv to PG16 IgG yielded a bispecific reagent whose neutralization activity combined activities from both parent antibodies. Although the neutralization activity due to each component was partially reduced, the combined reagent is attractive since fewer strains escaped neutralization.
Antibodies against the CD4 binding site (CD4bs) on the HIV-1 spike protein gp120 can show exceptional potency and breadth. We determined structures of NIH45-46, a more potent clonal variant of VRC01, alone and bound to gp120. Comparisons with VRC01–gp120 revealed that a four-residue insertion in CDRH3 contributed to increased interaction between NIH45-46 and the gp120 inner domain, which correlated with enhanced neutralization. We used structure-based design to create NIH45-46G54W, a single substitution in CDRH2 that increases contact with the gp120 bridging sheet and improves breadth and potency, critical properties for potential clinical use, by an order of magnitude. Together with the NIH45-46–gp120 structure, these results indicate that gp120 inner domain/bridging sheet residues should be included in immunogens to elicit CD4bs antibodies.
There is clearly a necessity to identify novel non-dopaminergic mechanisms as new therapeutic targets for Parkinson's disease (PD). Among these, the soluble guanylyl cyclase (sGC)-cGMP signaling cascade is emerging as a promising candidate for second messenger-based therapies for the amelioration of PD symptoms. In the present study, we examined the utility of the selective sGC inhibitor 1H-, ,  oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) for reversing basal ganglia dysfunction and akinesia in animal models of PD.
The utility of the selective sGC inhibitor ODQ for reversing biochemical, electrophysiological, histochemical, and behavioral correlates of experimental PD was performed in 6-OHDA-lesioned rats and mice chronically treated with MPTP.
We found that one systemic administration of ODQ is sufficient to reverse the characteristic elevations in striatal cGMP levels, striatal output neuron activity, and metabolic activity in the subthalamic nucleus observed in 6-OHDA-lesioned rats. The latter outcome was reproduced after intrastriatal infusion of ODQ. Systemic administration of ODQ was also effective in improving deficits in forelimb akinesia induced by 6-OHDA and MPTP.
Pharmacological inhibition of the sGC-cGMP signaling pathway is a promising non-dopaminergic treatment strategy for restoring basal ganglia dysfunction and attenuating motor symptoms associated with PD.
Neogenin is a type I transmembrane glycoprotein with a large ectodomain containing tandem immunoglobulin-like and fibronectin type III (FNIII) domains. Closely related to the tumor suppressor gene DCC, neogenin functions in critical biological processes through binding to various ligands, including netrin, repulsive guidance molecules, and the iron regulatory protein hemojuvelin. We previously reported that neogenin binds to hemojuvelin through its membrane-proximal fifth and sixth FNIII domains (FN5-6), with domain 6 (FN6) contributing the majority of critical binding interactions. Here we present the crystal structure of FN5-6, the hemojuvelin-binding fragment of human neogenin, at 1.8 Å. The two FNIII domains are orientated nearly linearly, a domain arrangement most similar to that of a tandem FNIII-containing fragment within the cytoplasmic tail of the β4 integrin. By mapping surface-exposed residues that differ between neogenin FN5-6 and the comparable domains from DCC, which does not bind hemojuvelin, we identified a potential hemojuvelin binding site on neogenin FN6. Neogenin FN5, which does not bind hemojuvelin in isolation, exhibits a highly electropositive surface, which may be involved in interactions with negatively-charged polysaccharides or phospholipids in the membrane bilayer. The neogenin FN5-6 structure can be used to facilitate a molecular understanding of neogenin’s interaction with hemojuvelin to regulate iron homeostasis and with hemojuvelin-related repulsive guidance molecules to mediate axon guidance.
neogenin; hemojuvelin; crystal structure; FNIII domain; iron homeostasis; repulsive guidance molecule
We present the first genome sequence of Chlamydophila psittaci, an intracellular pathogen of birds and a human zoonotic pathogen. A comparison with previously sequenced Chlamydophila genomes shows that, as in other chlamydiae, most of the genome diversity is restricted to the plasticity zone. The C. psittaci plasmid was also sequenced.
Striatal nitric oxide (NO)-producing interneurons play an important role in the regulation of corticostriatal synaptic transmission and motor behavior. Striatal NO synthesis is driven by concurrent activation of NMDA and dopamine (DA) D1 receptors. NO diffuses into the dendrites of medium-sized spiny neurons which contain high levels of NO receptors called soluble guanylyl cyclases (sGC). NO-mediated activation of sGC leads to the synthesis of the second messenger cGMP. In the intact striatum, transient elevations in intracellular cGMP primarily act to increase neuronal excitability and to facilitate glutamatergic corticostriatal transmission. NO–cGMP signaling also functionally opposes the inhibitory effects of DA D2 receptor activation on corticostriatal transmission. Not surprisingly, abnormal striatal NO–sGC–cGMP signaling becomes apparent following striatal DA depletion, an alteration thought to contribute to pathophysiological changes observed in basal ganglia circuits in Parkinson's disease (PD). Here, we discuss recent developments in the field which have shed light on the role of NO–sGC–cGMP signaling pathways in basal ganglia dysfunction and motor symptoms associated with PD and l-DOPA-induced dyskinesias.
basal ganglia; striatum; dopamine; nitric oxide; Parkinson's disease
We previously showed that broadly neutralizing anti-HIV-1 antibody 2G12 (human IgG1) naturally forms dimers that are more potent than monomeric 2G12 in in vitro neutralization of various strains of HIV-1. In this study, we have investigated the protective effects of monomeric versus dimeric 2G12 against HIV-1 infection in vivo using a humanized mouse model. Our results showed that passively transferred, purified 2G12 dimer is more potent than 2G12 monomer at preventing CD4 T cell loss and suppressing the increase of viral load following HIV-1 infection of humanized mice. Using humanized mice bearing IgG “backpack” tumors that provided 2G12 antibodies continuously, we found that a sustained dimer concentration of 5–25 µg/ml during the course of infection provides effective protection against HIV-1. Importantly, 2G12 dimer at this concentration does not favor mutations of the HIV-1 envelope that would cause the virus to completely escape 2G12 neutralization. We have therefore identified dimeric 2G12 as a potent prophylactic reagent against HIV-1 in vivo, which could be used as part of an antibody cocktail to prevent HIV-1 infection.
Most successful vaccines function by eliciting antibodies that bind to the surface of pathogens of interest from the host immunologic repertoire. This should also be the case for an HIV-1 vaccine, but broadly neutralizing anti-HIV-1 antibodies have proven hard to elicit with any reagent. Thus, methods to directly administer broadly neutralizing anti-HIV-1 antibodies, such as passive transfusion, become appealing. It is therefore important to find out which antibodies, or antibody cocktails, would provide effective protection against HIV-1 before administering them. Here, we show that the dimeric fraction of 2G12, a unique monoclonal anti-HIV-1 antibody that dimerizes naturally, provides better protection against HIV-1 than its monomeric fraction. As an added bonus, although HIV-1 can evolve to completely escape antibody control, the 2G12 dimer does not favor such evolution. Our study suggests that the 2G12 dimer may be a suitable reagent for direct administration to protect people from HIV-1 infection.
The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) has several adaptations that allow the virus to evade antibody neutralization. Nevertheless, a few broadly cross-reactive neutralizing antibodies as well as reagents containing portions of CD4, the HIV receptor, have demonstrated partial efficacy in suppressing viral replication. One type of reagent designed for improved HIV neutralization fuses the CD4 D1-D2 domains to the variable regions of an antibody recognizing the CD4-induced (CD4i) coreceptor binding site on the gp120 portion of the HIV envelope spike. We designed, expressed, purified, and tested the neutralization potencies of CD4-CD4i antibody reagents with different architectures, antibody combining sites, and linkers. We found that fusing CD4 to the heavy chain of the CD4i antibody E51 yields a bivalent reagent including an antibody Fc region that expresses well, is expected to have a long serum half-life, and has comparable or greater neutralization activity than well-known broadly neutralizing anti-HIV antibodies. A CD4 fusion with the anti-HIV carbohydrate antibody 2G12 also results in a potent neutralizing reagent with more broadly neutralizing activity than 2G12 alone.
The poxvirus 2L protein binds tumor necrosis factor-α (TNFα) to inhibit host antiviral and immune responses. The 2.8-Å 2L–TNFα structure reveals three symmetrically arranged 2L molecules per TNFα trimer. 2L resembles class I major histocompatibility complex (MHC) molecules but lacks a peptide-binding groove and β2-microglobulin light chain. Overlap between the 2L and host TNF receptor-binding sites on TNFα rationalizes 2L inhibition of TNFα–TNF receptor interactions and prevention of TNFα-induced immune responses.
Striatal medium-sized spiny neurons (MSNs) contain the highest levels of soluble guanylyl cyclase (sGC) in the brain. Striatal sGC signaling is activated by nitric oxide (NO) and other neuromodulators. MSNs also express cGMP-dependent protein kinase and other components of the cGMP signaling system which are critically involved in integrating corticostriatal transmission and regulating synaptic plasticity in striatal networks. However, the influence of tonic and phasic activation of this signaling pathway on striatal MSN activity is poorly understood. The present study examined the impact of systemic administration of the selective sGC inhibitor [1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) on spike activity evoked using low and high frequency electrical stimulation of the frontal cortex. MSN activity was monitored using single-unit extracellular recordings in urethane-anesthetized rats. ODQ administration significantly decreased spike activity evoked by low frequency cortical stimulation in a stimulus intensity- and time-dependent manner. Additionally, ODQ administered along with the neuronal NO synthase inhibitor 7-nitroindazole (7-NI) potently decreased the incidence of excitatory responses observed during high frequency train stimulation of the contralateral frontal cortex. The short-term depression of cortically-evoked spike activity induced by train stimulation was enhanced following pretreatment with ODQ in MSNs exhibiting an excitatory response during cortical train stimulation. Unexpectedly, this effect of ODQ was reversed in animals receiving co-administration of ODQ and 7-NI. 7-NI/ODQ co-administration also reversed measures of short-term depression observed in MSNs exhibiting an inhibitory response during cortical train stimulation. These observations extend previous studies showing that tonic and phasic NO-sGC signaling modulates the responsiveness of MSNs to corticostriatal input. Moreover, phasic activation of NO signaling is likely to regulate short-term changes in corticostriatal synaptic plasticity via complex mechanisms involving both sGC-cGMP-dependent and independent pathways.
striatum; nitric oxide synthase; cyclic nucleotides; short-term depression; electrophysiology
Hemojuvelin is a recently-identified iron-regulatory protein that plays an important role in affecting the expression of hepcidin, a key iron regulatory hormone. Although the underlying mechanism of this process is not clear, several hemojuvelin-binding proteins, including the cell surface receptor neogenin and bone morphogenetic protein (BMP) cytokines, have been identified. The ectodomain of neogenin is composed of four immunoglobulin-like (Ig) domains followed by six fibronectin type III-like (FNIII) domains. Here we report expression of soluble versions of hemojuvelin and neogenin for biochemical characterization of their interaction and the interaction of HJV with BMP-2. Hemojuvelin normally undergoes an autocatalytic cleavage, and as in vivo, recombinant hemojuvelin exists as a mixture of cleaved and uncleaved forms. Neogenin binds to cleaved and non-cleaved hemojuvelin, as verified by its binding to an uncleaved mutant hemojuvelin. We localized the hemojuvelin binding site on neogenin to the membrane-proximal fifth and sixth FNIII domains and the juxtamembrane linker, and showed that a fragment containing only this region binds 2-3 orders of magnitude more tightly than the entire neogenin ectodomain. Binding to the most membrane-proximal region of neogenin may play a role in regulating the levels of soluble and membrane-bound forms of hemojuvelin, which in turn would influence the amount of free BMP-2 available for binding to its receptors and triggering transcription of the hepcidin gene. Our finding that BMP-2 and neogenin bind simultaneously to hemojuvelin raises the possibility that neogenin is part of a multi-protein complex at the hepatocyte membrane involving BMP, its receptors, and hemojuvelin.
It is known that dopamine (DA) D1 receptor activation stimulates striatal nitric oxide (NO) synthesis, whereas D2 receptor activation produces the opposite effect. However, the mechanisms involved in the dopaminergic modulation of NO synthase (NOS) are unknown.
We hypothesized that the effects of DA on striatal NO signaling are dependent on ongoing glutamatergic activation of NOS. Therefore, the current study examined whether intact NMDA receptor activation is required for the dopaminergic modulation of NOS activity.
We assessed the impact of pharmacological manipulations of D1, D2 and NMDA receptors on NOS activity in the dorsal striatum and motor cortex using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Drugs were administered systemically to conscious animals and NADPH-d staining was quantified in these regions using ex vivo measurements of tissue optical density.
Administration of the neuronal NOS inhibitor NG-propyl-L-arginine (NPA), the D1 receptor antagonist SCH 23390, and the NMDA receptor antagonist 3-phosphonopropyl-piperazine-2-carboxylic acid (CPP) all attenuated staining selectively in the striatum. Administration of the D2 receptor agonist quinpirole decreased NADPH-d staining in both the striatum and cortex. Striatal NADPH-d staining elicited by administration of the D1 receptor agonist SKF 81297 or the D2 receptor antagonist eticlopride was attenuated by NPA, SCH 23390, and CPP pretreatment. Quinpirole pretreatment also abolished the facilitatory effect of SKF 81297.
These studies show for the first time that ongoing NMDA receptor activation is necessary for modulation of striatal NOS activity by both facilitatory (D1 receptor activation) and inhibitory (D2 receptor activation) dopaminergic signaling mechanisms.
dopamine; nitric oxide; nitric oxide synthase; NMDA receptor; striatum; cortex
G protein-coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins1. Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains2. Down-regulation of mth increases the lifespan of Drosophila melanogaster3— inhibitors of Mth signaling would thus be expected to enhance longevity. We used mRNA display selection4,5 to identify high affinity (KD = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane (TM) domains. Flies constitutively expressing a Mth antagonist peptide exhibit a robust lifespan extension, suggesting that the peptides inhibit Mth signaling in vivo. Our work thus provides novel lifespan-extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.
We have previously shown that cocaine enhances gene regulation in the sensorimotor striatum associated with procedural learning in a running-wheel paradigm. Here we assessed whether cocaine produces enduring modifications of learning-related changes in striatal neuron activity, using single-unit recordings in anesthetized rats 1 day after the wheel training. Spontaneous and cortically evoked spike activity was compared between groups treated with cocaine or vehicle immediately prior to the running-wheel training or placement in a locked wheel (control conditions). We found that wheel training in vehicle-treated rats increased the average firing rate of spontaneously active neurons without changing the relative proportion of active to quiescent cells. In contrast, in rats trained under the influence of cocaine, the proportion of spontaneously firing to quiescent cells was significantly greater than in vehicle-treated, trained rats. However, this effect was associated with a lower average firing rate in these spontaneously active cells, suggesting that training under the influence of cocaine recruited additional low-firing cells. Measures of cortically evoked activity revealed a second interaction between cocaine treatment and wheel training, namely, a cocaine-induced decrease in spike onset latency in control rats (locked wheel). This facilitatory effect of cocaine was abolished when rats trained in the running wheel during cocaine action. These findings highlight important interactions between cocaine and procedural learning, which act to modify population firing activity and the responsiveness of striatal neurons to excitatory inputs. Moreover, these effects were found 24 h after the training and last drug exposure indicating that cocaine exposure during the learning phase triggers long-lasting changes in synaptic plasticity in the dorsal striatum. Such changes may contribute to the transition from recreational to habitual or compulsive drug taking behavior.
corticostriatal transmission; dorsal striatum; medium-sized spiny neuron; habit formation; procedural learning; addiction
Nitric oxide (NO) is a key neuromodulator of corticostriatal synaptic transmission. We have shown previously that dopamine (DA) D1/5 receptor stimulation facilitates neuronal NO synthase (nNOS) activity in the intact striatum. To study the impact of local manipulations of D1/5 and glutamatergic NMDA receptors on striatal nNOS activity, we combined the techniques of in vivo amperometry and reverse microdialysis. Striatal NO efflux was monitored proximal to the microdialysis probe in urethane anesthetized rats during local infusion of vehicle or drug. NO efflux elicited by systemic administration of SKF-81297 was blocked following intrastriatal infusion of: 1) the D1/5 receptor antagonist SCH-23390, 2) the nNOS inhibitor 7-nitroindazole, 3) the nonspecific ionotropic glutamate receptor antagonist kynurenic acid, and 4) the selective NMDA receptor antagonist 3-phosphonopropyl-piperazine-2-carboxylic acid. Glycine coperfusion did not affect SKF-81297-induced NO efflux. Furthermore, intrastriatal infusion of SKF-81297 potentiated NO efflux evoked during electrical stimulation of the motor cortex. The facilitatory effects of cortical stimulation and SKF-81297 were both blocked by intrastriatal infusion of SCH-23390, indicating that striatal D1/5 receptor activation is necessary for the activation of nNOS by corticostriatal afferents. These studies demonstrate for the first time that reciprocal DA-glutamate interactions play a critical role in stimulating striatal nNOS activity.
Nitric oxide; Glutamate; Dopamine; D1/5 receptor; Dorsal striatum; NMDA receptor; Neuronal nitric oxide synthase
The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.
The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.