Resting Hypoconnectivity of Theoretically Defined Addiction Networks during Early Abstinence Predicts Subsequent Relapse in Alcohol Use Disorder.

Theoretical models of addiction suggest that alterations in addiction domains including incentive salience, negative emotionality, and executive control lead to relapse in alcohol use disorder (AUD). To determine whether the functional organization of neural networks underlying these domains predict subsequent relapse, we generated theoretically defined addiction networks. We collected resting functional magnetic resonance imaging data from 45 individuals with AUD during early abstinence (number of days abstinent M = 25.40, SD = 16.51) and calculated the degree of resting-state functional connectivity (RSFC) within these networks. Regression analyses determined whether the RSFC strength in domain-defined addiction networks measured during early abstinence predicted subsequent relapse (dichotomous or continuous relapse metrics). RSFC within each addiction network measured during early abstinence was significantly lower in those that relapsed (vs. abstained) and predicted subsequent time to relapse. Lower incentive salience RSFC during early abstinence increased the odds of relapsing. Neither RSFC in a control network nor clinical self-report measures predicted relapse. The association between low incentive salience RSFC and faster relapse highlights the need to design timely interventions that enhance RSFC in AUD individuals at risk of relapsing faster.

Exploratory study of how Cognitive Multisensory Rehabilitation restores parietal operculum connectivity and improves upper limb movements in chronic stroke.

Cognitive Multisensory Rehabilitation (CMR) is a promising therapy for upper limb recovery in stroke, but the brain mechanisms are unknown. We previously demonstrated that the parietal operculum (parts OP1/OP4) is activated with CMR exercises. In this exploratory study, we assessed the baseline difference between OP1/OP4 functional connectivity (FC) at rest in stroke versus healthy adults to then explore whether CMR affects OP1/OP4 connectivity and sensorimotor recovery after stroke. We recruited 8 adults with chronic stroke and left hemiplegia/paresis and 22 healthy adults. Resting-state FC with the OP1/OP4 region-of-interest in the affected hemisphere was analysed before and after 6 weeks of CMR. We evaluated sensorimotor function and activities of daily life pre- and post-CMR, and at 1-year post-CMR. At baseline, we found decreased FC between the right OP1/OP4 and 34 areas distributed across all lobes in stroke versus healthy adults. After CMR, only four areas had decreased FC compared to healthy adults. Compared to baseline (pre-CMR), participants improved on motor function (MESUPES arm p = 0.02; MESUPES hand p = 0.03; MESUPES total score p = 0.006); on stereognosis (p = 0.03); and on the Frenchay Activities Index (p = 0.03) at post-CMR and at 1-year follow-up. These results suggest enhanced sensorimotor recovery post-stroke after CMR. Our results justify larger-scale studies.

Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis.

gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.

Defining mechanisms of toxicity for linoleic acid monoepoxides and diols in Sf-21 cells.

Linoleic acid monoepoxides have been correlated with many pathological conditions. Studies using insect cells derived from Spodoptera frugiperda (Sf-21 cells) have suggested that conversion of the epoxides to the diols is required for toxicity. However, more recent studies using rabbit renal proximal tubules have suggested that linoleic acid monoepoxides are direct mitochondrial toxins. To better understand these discrepancies, we compared the toxicity of these linoleic acid metabolites in Sf-21 cells using mitochondrial respiration as an end point. Linoleic acid (100 microM) and 12,13-epoxy-9-octadecenoic acid (12,13-EOA, 100 microM) increased the rate of oligomycin-insensitive respiration by approximately 3.5- and 3-fold, respectively, decreased the rate of oligomycin-sensitive respiration by approximately 52 and 68%, respectively, and had no effect on the integrity of the electron transport chain. These effects were concentration-dependent, occurred within 1 min, and recovered to basal levels within 45 min. 12,13-Dihydroxy-9-octadecenoic acid (12,13-DHOA, 100 microM) had no effect on oligomycin-insensitive respiration but decreased the rate of oligomycin-sensitive respiration and uncoupled respiration in a concentration-dependent manner. Approximately 79 and 68% of oligomycin-sensitive respiration and uncoupled respiration was inhibited by 12,13-DHOA (100 microM), respectively. These effects occurred within 1 min and were not reversible in 6 h. Effects similar to those induced by 12,13-DHOA (100 microM) were observed using 12,13-EOA (100 microM) in Sf-21 cells expressing human soluble epoxide hydrolase. These data suggest that in this Sf-21 model linoleic acid and linoleic monoepoxides have transient uncoupling effects, whereas the primary mechanism of toxicity for linoleic acid diols in this model is inhibition of the electron transport chain.

Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway.

Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs. For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site. Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome. All mutations obtained were located in the editing site of valyl-tRNA synthetase. More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine. Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids. Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation.

Errors from selective disruption of the editing center in a tRNA synthetase.

Some aminoacyl-tRNA synthetases have two catalytic centers that together achieve fine-structure discrimination of closely similar amino acids. The role of tRNA is to stimulate translocation of a misactivated amino acid from the active site to the editing site where the misactivated substrate is eliminated by hydrolysis. Using isoleucyl-tRNA synthetase as an example, we placed mutations in the catalytic center for editing at residues strongly conserved from bacteria to humans. A particular single substitution and one double substitution resulted in production of mischarged tRNA, by interfering specifically with the chemical step of hydrolytic editing. The substitutions affected neither amino acid activation nor aminoacylation, with the cognate amino acid. Thus, because of the demonstrated functional independence of the two catalytic sites, errors of aminoacylation can be generated by selective mutations in the center for editing.

Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis.

Misactivation of amino acids by aminoacyl-tRNA synthetases can lead to significant errors in protein synthesis that are prevented by editing reactions. As an example, discrete sites in isoleucyl-tRNA synthetase for amino acid activation and editing are about 25 A apart. The details of how misactivated valine is translocated from one site to the other are unknown. Here, we present a kinetic study in which a fluorescent probe is used to monitor translocation of misactivated valine from the active site to the editing site. Isoleucine-specific tRNA, and not other tRNAs, is essential for translocation of misactivated valine. Misactivation and translocation occur on the same enzyme molecule, with translocation being rate limiting for editing. These results illustrate a remarkable capacity for a specific tRNA to enhance amino acid fine structure recognition by triggering a unimolecular translocation event.

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.

Antibody transport in cultured tumor cell layers.

This review summarizes our recent in vitro studies of the factors affecting the tumor penetration of immunoconjugates. The studies were designed to probe the mechanisms of diffusion and convection, using a cultured layer of mouse melanoma cells as a model tumor cell layer and an antibody to the murine transferrin receptor as a model ligand. Transport of the binding antibody was observed to be slower than that of a non-binding control, a result that is consistent with the "binding site barrier" hypothesis (Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Internalization of the antibody/receptor complex was necessary for this effect to be observed, implying that intracellular trafficking is a determinant of net tumor transport rates. Convective fluid flow exhibited a dependence on cell density that is consistent with a Poiseuille flow model, suggesting that convective transport occurs as laminar flow in tortuous channels. Implications for immunoconjugate therapy, limitations of the approach, and future directions of the research program are discussed.

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements.

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.

Postdialysis fatigue: lack of effect of a biocompatible membrane.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to have somnogenic properties. Plasma levels of this cytokine have been found to increase significantly during dialysis with a bioincompatible (cuprophane) membrane in patients with postdialysis fatigue (PDF). We conducted a crossover study with random assignment to ascertain whether a biocompatible membrane might attenuate the increase of TNF-alpha and severity of PDF. Sixteen patients on maintenance hemodialysis underwent dialysis with either cuprophane (n = 8) or polymethylmethacrylate (PMMA; n = 8) membranes for 1 week and then switched to the opposite membrane during the second week. Predialysis and postdialysis measurements of plasma TNF-alpha levels were performed during the first and last dialysis treatments of each week. A fatigue score was determined from the sum of duration of fatigue and sleep within 6 hours of the completion of dialysis. TNF-alpha levels increased by an average of 18.3% during dialysis with cuprophane membranes but only 2.4% with PMMA membranes (P = 0.04). Despite this, fatigue scores remained unaltered (approximately 4 of 6). Hence, the biocompatible membrane, PMMA, failed to alleviate PDF. This suggests that dialytic stimulation of TNF-alpha plays no substantial role in the pathogenesis of PDF.

Enzyme structure with two catalytic sites for double-sieve selection of substrate.

High-fidelity transfers of genetic information in the central dogma can be achieved by a reaction called editing. The crystal structure of an enzyme with editing activity in translation is presented here at 2.5 angstroms resolution. The enzyme, isoleucyl-transfer RNA synthetase, activates not only the cognate substrate L-isoleucine but also the minimally distinct L-valine in the first, aminoacylation step. Then, in a second, "editing" step, the synthetase itself rapidly hydrolyzes only the valylated products. For this two-step substrate selection, a "double-sieve" mechanism has already been proposed. The present crystal structures of the synthetase in complexes with L-isoleucine and L-valine demonstrate that the first sieve is on the aminoacylation domain containing the Rossmann fold, whereas the second, editing sieve exists on a globular beta-barrel domain that protrudes from the aminoacylation domain.

Elevated levels of tumor necrosis factor alpha in postdialysis fatigue.

Postdialysis fatigue (PDF) has been ascribed to excessive ultrafiltration and decline in osmolality during hemodialysis. We evaluated the potential role for the sommogenic cytokines, interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), in the genesis of PDF Patients dialyzing with cuprophane membrane were assigned to PDF (N=25) and non-PDF (N=25) groups based on a fatigue index questionnaire. Pre- and postdialysis samples were obtained from 3 consecutive treatments and later assayed for serum levels of IL-1beta and TNFalpha by ELISA. Our results show significant intradialytic elevation of TNFalpha in both non-PDF groups (non-PDF: pre- 3.36+/-0.80 pg/ml to post 3.75+/-0.88 pg/ml, p<0.04; PDF: pre- 5.95+/-0.80 pg/ml to post- 8.66-/+1.35 pg/ml, p<0.02). The degree of intradialytic augmentation was significantly greater for TNFalpha in the PDF group (46+/-18% vs 11+/-5%; p<0.03). There were no significant intradialytic changes in serum levels of IL-1beta in either the PDF or non-PDF groups. There also were no significant differences in dialysis-related body weights, systolic blood pressures, or osmolalities. These findings suggest that TNFalpha may be involved in the pathogenesis of PDF.

The psychiatric consultant in professional team sports.

The role of the sport psychiatrist consulting to a professional sports team is complex, challenging, and varied. Sport psychiatrists enhance the psychosocial development of the athlete by removing obstacles to growth and by facilitating lines of communication that support exchange of information, sharing of feelings, and the development of solid winning relationships on the team and throughout the organization. The consultant psychiatrist often provides input both to players and to management, however; these dual responsibilities raise ethical concerns and often require the services of additional treating psychiatrists and other mental health professionals.

Structural and functional analysis of peptidyl oligosaccharyl transferase inhibitors.

The peptide cyclo(hex-Amb(1)-Cys(2))-Thr(3)-Val(4)-Thr(5)-Nph(6)-NH2 was previously shown to be a slow, tight-binding inhibitor (Ki = 37 nM) of the yeast oligosaccharyl transferase (OT) [Hendrickson et al. (1996) J. Am. Chem. Soc. 118, 7636-7637]. This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus sequence Asn-Xaa-Thr/Ser. Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding. Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation of an additional methylene into the side chain of residue 1 [(S)-2,3-diaminobutyric acid changed to ornithine] had very little effect. Variants with acidic, basic, hydrophilic/polar, and hydrophobic side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition. This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (threonine) residues are both well tolerated. The kinetic behavior of substrate analogs [cyclo(hex-Asn(1)-Cys(2))-Thr(3)-Xaa(4)-Yaa(5)-Nph-NH2] corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors.