Continuous Sensory-Motor Responses in Humans

A vast amount of psychological and physiological data has been accumulated on human responses as elementary models of human behavior. The dynamic structure of human responses in time and the characteristics of their sensory and motor components have been described in detail, and their dependence on a wide variety of factors has been revealed. Diverse aspects of the mechanism regulating human motor responses have been studied. (1)     

Sensory-Motor Interactions for Vocal Pitch Monitoring in Non-Primary Human Auditory Cortex

The neural mechanisms underlying processing of auditory feedback during self-vocalization are poorly understood. One technique used to study the role of auditory feedback involves shifting the pitch of the feedback that a speaker receives, known as pitch-shifted feedback. We utilized a pitch shift self-vocalization and playback paradigm to investigate the underlying neural mechanisms of audio-vocal interaction. High-resolution electrocorticography (ECoG) signals were recorded directly from auditory cortex of 10 human subjects while they vocalized and received brief downward (−100 cents) pitch perturbations in their voice auditory feedback (speaking task). ECoG was also recorded when subjects passively listened to playback of their own pitch-shifted vocalizations. Feedback pitch perturbations elicited average evoked potential (AEP) and event-related band power (ERBP) responses, primarily in the high gamma (70–150 Hz) range, in focal areas of non-primary auditory cortex on superior temporal gyrus (STG). The AEPs and high gamma responses were both modulated by speaking compared with playback in a subset of STG contacts. From these contacts, a majority showed significant enhancement of high gamma power and AEP responses during speaking while the remaining contacts showed attenuated response amplitudes. The speaking-induced enhancement effect suggests that engaging the vocal motor system can modulate auditory cortical processing of self-produced sounds in such a way as to increase neural sensitivity for feedback pitch error detection. It is likely that mechanisms such as efference copies may be involved in this process, and modulation of AEP and high gamma responses imply that such modulatory effects may affect different cortical generators within distinctive functional networks that drive voice production and control.     

A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies

The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W⁶⁷⁰NWFDITN⁶⁷⁷) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens.The continued spread of human immunodeficiency virus (HIV) worldwide and, in particular, in sub-Saharan Africa, where an estimated 22 million people currently are living with HIV/AIDS, underscores the urgent need for a preventative vaccine. However, despite nearly 25 years of intense international research, a vaccine is not yet available. Passive immunization with broadly neutralizing antibodies can confer sterilizing protection against infection in animal models (4, 12, 39-41, 51, 64), providing encouragement for the development of an antibody-inducing component of an HIV type 1 (HIV-1) vaccine. Such a vaccine should elicit neutralizing antibodies with activity against the broadest range of primary circulating isolates. However, a lack of understanding of how to raise potent, cross-reactive antibodies by immunization, the so-called neutralizing antibody problem, is a major hurdle in this effort (6, 24, 72). Thus, an understanding of the structure and presentation of neutralizing epitopes on the virus and the antibodies that recognize them is vital for vaccine development.The targets of antibody neutralization are the surface envelope (Env) glycoprotein trimers (gp120/gp41) that mediate the fusion of the viral membrane with that of the host. The majority of antibodies elicited during natural infection or immunization show limited or no cross-reactivity against diverse isolates. However, a few rare, broadly neutralizing, monoclonal antibodies have been isolated from HIV-1-infected individuals and exhibit activity against a wide range of isolates by binding to functionally conserved epitopes exposed on native gp120/gp41 trimers. These epitopes include the CD4 binding site, recognized by antibody b12, and a relatively well-conserved cluster of N-linked glycans, located on the outer domain of gp120, that is recognized by antibody 2G12 (12, 13, 71, 76). V3-directed antibodies, which are common in natural infection, also are able to sporadically neutralize across clades, as exemplified by 447-52D and F425-B4e8 (7, 16, 49, 66). The identification of three broadly neutralizing antibodies, 2F5, Z13, and 4E10, that target the conserved tryptophan-rich membrane-proximal external region (MPER) of gp41 has implicated this region as a highly promising vaccine target and has, therefore, spurred interest in its structural characterization (15, 35, 45, 47, 48, 50, 80).The MPER plays a critical, but not fully understood, role in membrane fusion and is situated between the C-terminal heptad repeat (CHR) and the transmembrane domain (TM) of gp41 (Fig. ​(Fig.1).1). Following the binding of gp120 to the cell surface receptors CD4 and CXCR4/CCR5, the gp41 glycoprotein undergoes a series of conformational changes that trigger the membrane fusion activity. Notably, a relatively long-lived prehairpin intermediate of gp41 is formed, in which the coiled-coil of the N-terminal heptad repeats (NHR) extends so as to enable the fusion peptides to embed into the target membrane. In the postfusion or fusogenic state, the CHR and NHR reassemble into an antiparallel 6-helix bundle in a process that drives membrane fusion (18). The MPER contains several functionally conserved tryptophan residues that are critical for membrane fusion and viral entry, although the structural basis for their specific role has not been firmly established (22, 44, 58). Their mutation to alanine leads to the attenuation of viral infectivity, which is most pronounced for Trp666 and Trp672 (numbered according to the HXB2 isolate) (46, 58, 78). In addition, peptides based on the MPER can induce membrane leakage (68). Such membrane-disrupting properties of the MPER have been suggested to be functionally important in the expansion of the fusion pore created after receptor engagement (42, 44, 58, 68, 77).Open in a separate windowFIG. 1.Major features of gp41 include the fusion peptide (FP), NHR, CHR, TM, and cytoplasmic domain (CD). The MPER is located between the CHR and TM regions of gp41. The core epitopes of 2F5 (green), Z13e1 (yellow), and 4E10 (orange) are indicated. The epitope of Z13e1 is located between those of 2F5 and 4E10, but it overlaps more closely with 4E10.From initial explorations using solution nuclear magnetic resonance, the structure of a 19-residue MPER peptide (residues 665 to 683) was found to be helical in dodecylphosphocholine micelles, with the hydrophobic and hydrophilic residues distributed evenly around the helix axis (62). Another study found that an MPER peptide comprising residues 659 to 671 adopts a 3₁₀-helix in water (10). More recently, the structure of an MPER peptide (residues 662 to 683) in liposomes was elucidated by a combination of nuclear magnetic resonance and spin-label electron paramagnetic resonance (69), and it was found to adopt a kinked, amphipathic structure composed of two helices connected by a short hinge (Phe673 and Asn674). Crystal structures of Fab 2F5 in complex with a 7-mer (E⁶⁶²LDKWAS⁶⁶⁸) and 17-mer encompassing residues 654 to 670 previously had revealed a mostly extended conformation characterized by a central β-turn involving Asp664, Lys665, and Trp666 (47, 48). This motif is the key recognition determinant for 2F5 and becomes deeply buried in the antibody combining site, suggesting that it is exposed at some stage in viral entry (45, 47, 78). The crystal structure of Fab 4E10 in complex with peptide-spanning residues W⁶⁷⁰NWFDITNW⁶⁷⁸ revealed an amphipathic α-helical structure with a narrow hydrophilic face (15). The N terminus of the 4E10 epitope forms a 3₁₀-helix that transitions into a regular α-helix at residue Asp674 and continues to Lys683, which constitutes the end of the gp41 ectodomain (14). Thus, while the structure of the MPER within functional, membrane-embedded Env trimers is not known, the observation that unconstrained peptides are able to adopt more than one defined structure suggests an inherent degree of flexibility.Like 4E10, Z13 was identified from an HIV-1-infected individual, the former being isolated from an immortalized B-cell line and the latter from a bone marrow RNA phage display library (80). The epitope of MAb Z13 spans residues S⁶⁶⁸LWNWFDITN⁶⁷⁷, as determined by peptide mapping, scanning mutagenesis, and antibody competition studies (46, 80). This region lies between the 2F5 and 4E10 epitopes but overlaps more closely with 4E10 (Fig. ​(Fig.1).1). 4E10 and Z13 are both able to neutralize primary as well as laboratory-adapted isolates; nevertheless, Z13 is not as broadly neutralizing as 4E10, which has the greatest breadth of any HIV-1 antibody described to date (9). Z13e1 is an affinity-enhanced variant of Z13 and was evolved by randomizing the complementarity determining region (CDR) L3 loop sequence to identify tighter-binding mutants using phage display (46). Z13e1 displays higher affinity for both peptide and recombinant gp41 substrates, as well as increased neutralization potency, suggesting that the L3 mutations optimize binding to the linear MPER epitope. The neutralization breadth of Z13e1 is limited by the requirement for Asn671 and Asp674 in the MPER, which are approximately 71 and 58% conserved, respectively, among sequences in the Los Alamos HIV sequence database (80). Based on the clear relationship between Env trimer binding and neutralization, the neutralizing activity of Z13e1 derives from binding to a functional trimer (8, 20, 25, 43, 52, 55, 60, 73, 74). While Z13e1 and 4E10 have identical affinities for optimized linear peptides, Z13e1 is still about an order of magnitude less potent than 4E10 against a variety of primary isolates. Although the occlusion of the Z13e1 epitope on virion-associated trimers is thought to be the major limitation (46), the structural basis for the lower potency of Z13e1 relative to those of 2F5 and 4E10 is unclear.Whereas neutralization by 4E10 depends critically on Trp672 and Phe673, Z13e1 instead requires the flanking Asn671 and Asp674 residues (46). Based on a helical model of the MPER, it was predicted that Z13e1 binds the narrow hydrophilic face that displays Asn671, Asp674, and Asn677 that is opposite that recognized by 4E10. As Z13e1 and 4E10 bind to functional trimers, both epitopes must be exposed at some stage before membrane fusion (20). To examine how Z13e1 recognizes its MPER epitope, we determined the crystal structure of Fab Z13e1 in complex with a 12-residue peptide corresponding to the core epitope with C-terminal flanking lysines to aid peptide solubility (W⁶⁷⁰NWFDITN⁶⁷⁷KKKK). The crystal structure at 1.8-Å resolution uncovers a conformation of the MPER that is distinct from that visualized in complex with 4E10. Our findings show that Z13e1 and 4E10 recognize different conformers of the MPER and reveal a novel conformational switch that is relevant for HIV-1 neutralization and membrane fusion.     

Overlapping Messages and Survivability

The phenomenon of overlapping of various sequence messages in genomes is a puzzle for evolutionary theoreticians, geneticists, and sequence researchers. The overlapping is possible due to degeneracy of the messages, in particular, degeneracy of codons. It is often observed in organisms with a limited size of genome, possessing polymerases of low fidelity. The most accepted view considers the overlapping as a mechanism to increase the amount of information per unit length. Here we present a model that suggests direct evolutionary advantage of the message overlapping. Two opposing drives are considered: (a) reduction in the amount of vulnerable points when the overlapping of two messages involves common critical points and (b) cumulative compromising cost of coexistence of messages at the same site. Over a broad range of conditions the reduction of the target size prevails, thus making the overlapping of messages advantageous.[Reviewing Editor: Dr. Martin Kreitman]     

Detection of Signature Sequences in Overlapping Genes and Prediction of a Novel Overlapping Gene in Hepatitis G Virus

In viruses an increased coding ability is provided by overlapping genes, in which two alternative open reading frames (ORFs) may be translated to yield two distinct proteins. The identification of signature sequences in overlapping genes is a topic of particular interest, since additional out-of-frame coding regions can be nested within known genes. In this work, a novel feature peculiar to overlapping coding regions is presented. It was detected by analysis of a sample set of 21 virus genomic sequences and consisted in the repeated occurrence of a cluster of basic amino acid residues, encoded by a frame, combined to a stretch of acidic residues, encoded by the corresponding overlapping frame. A computer scan of an additional set of virus sequences demonstrated that this feature is common to several other known overlapping ORFs and led to prediction of a novel overlapping gene in hepatitis G virus (HGV). The occurrence of a bifunctional coding region in HGV was also supported by its extremely lower rate of synonymous nucleotide substitutions compared to that observed in the other gene regions of the HGV genome. Analysis of the amino acid sequence that was deduced from the putative overlapping gene revealed a high content of basic residues and the presence of a nuclear targeting signal; these characteristics suggest that a core-like protein may be expressed by this novel ORF. Received: 21 July 1999 / Accepted: 26 October 1999     

Structural Studies of Overlapping Dinucleosomes in Solution

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In this study, the solution structure of the OLDN was investigated through the integration of small-angle x-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis and searched for the conformations that reproduced the SAXS and SANS scattering curves well. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.     

Overlapping genes in parasitic protist Giardia lamblia.

The parasitic protist Giardia lamblia lacks mitochondria and peroxisomes, as well as many typical membrane-bound organella characteristics of higher eukaryotic cells, together with extremely economized usage of DNA sequence, as demonstrated by the lack of introns. We describe here the presence of overlapping genes in G. lamblia, in which a part of the protein coding sequence of one mRNA exists in a region corresponding to the 3′-noncoding region of another mRNA transcribed from a gene on the opposite strand. Recently we isolated 13 kinesin-related cDNAs from G. lamblia. Nine of these cDNAs contain long 3′-noncoding sequences in which long open reading frames (ORFs) exist (in the remaining four cDNAs, the lengths of the 3′-noncoding sequences are very short). The predicted amino acid sequences of these ORFs were subjected to a search for homologies with sequences in databases. The amino acid sequences of the six ORFs exhibited significant sequence similarities with known sequences. These lines of evidence suggest the frequent occurrence of gene overlap in Giardial genome.     

Overlapping Cardiac Programs in Heart Development and Regeneration

Gaining cellular and molecular insights into heart development and regeneration will likely provide new therapeutic targets and opportunities for cardiac regenerative medicine,one of the most urgent clinical needs for heart failure.Here we present a review on zebrafish heart development and regeneration,with a particular focus on early cardiac progenitor development and their contribution to building embryonic heart,as well as cellular and molecular programs in adult zebrafish heart regeneration.We attempt to emphasize that the signaling pathways shaping cardiac progenitors in heart development may also be redeployed during the progress of adult heart regeneration.A brief perspective highlights several important and promising research areas in this exciting field.     

Efficacy of Tricaine Methanesulfonate (MS-222) as an Anesthetic Agent for Blocking Sensory-Motor Responses in Xenopus laevis Tadpoles

Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222) is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes.     

Overlapping alternative donor splice sites in the human genome

Over 50% of donor splice sites in the human genome have a potential alternative donor site at a distance of three to six nucleotides. Conservation of these potential sites is determined by the consensus requirements and by its exonic or intronic location. Several hundred pairs of overlapping sites are confirmed to be alternatively spliced as both sites in a pair are supported by a protein, by a full-length mRNA, or by expressed sequence tags (ESTs) from at least two independent clone libraries. Overlapping sites may clash with consensus requirements. Pairs with a site shift of four nucleotides are the most abundant, despite the frameshift in the protein-coding region that they introduce. The site usage in pairs is usually uneven, and the major site is more frequently conserved in other mammalian genomes. Overlapping alternative donor sites and acceptor sites may have different functional roles: alternative splicing of overlapping acceptor sites leads mainly to microvariations in protein sequences; whereas alternative donor sites often lead to frameshifts and thus either yield major differences in the protein sequence and structure, or generate nonsense-mediated decay-inducing mRNA isoforms likely involved in regulated unproductive splicing pathways.     

Mining Dense Overlapping Subgraphs in weighted protein-protein interaction networks

Many methods have been proposed for mining protein complexes from a protein-protein interaction network; however, most of them focus on unweighted networks and cannot find overlapping protein complexes. Since one protein may serve different roles within different functional groups, mining overlapping protein complexes in a weighted protein-protein interaction network has attracted more and more attention recently. In this paper, we propose an effective method, called MDOS (Mining Dense Overlapping Subgraphs), for mining dense overlapping protein complexes (subgraphs) in a weighted protein-protein interaction network. The proposed method can integrate the information about known complexes into a weighted protein-protein interaction network to improve the mining results. The experiment results show that our method mines more known complexes and has higher sensitivity and accuracy than the CODENSE and MCL methods.