A change in the glutamatergic system is thought to play an important role in the pathophysiology of schizophrenia. The aim of this study was to investigate the changes in metabolites, including glutamate (Glu), in the anterior cingulate cortex (ACC) and the left basal ganglia (ltBG) of patients with chronic schizophrenia using proton magnetic resonance spectroscopy ((1)H-MRS). In addition, since gender differences in this illness were known, we examined the effects of gender on these metabolites. The (1)H-MRS was performed on the ACC and ltBG of 30 patients with schizophrenia and 25 healthy individuals who acted as the control group. The levels of Glu, glutamine (Gln), creatine plus phosphocreatine (Cre), myo-inositol (mI), N-acetylaspartate (NAA), and choline-containing compounds (Cho) were measured. Two-way analysis of variance revealed that the illness significantly affected the levels of Glu and mI in the ACC; both metabolites were lower in the patients with schizophrenia as compared to the control subjects. The results also revealed that gender significantly affected the level of Gln in the ACC and the levels of Cre and NAA in the ltBG; the level of Gln in the ACC were higher in male subjects versus female subjects, whereas Cre and NAA levels in the ltBG were lower in male subjects as compared to female subjects. These results confirmed a change in the glutamatergic system and suggested an involvement of mI in the pathophysiology of schizophrenia.

The higher plant Arabidopsis (Arabidopsis thaliana) has eight genes potentially coding for small ubiquitin-related modifier (SUMO) proteins. However, two well-expressed isoforms differ from fungal and animal consensus in a conserved glutamine (Gln) residue situated four residues from the carboxyl terminus. We tested deviations in this position in the background of SUMO1, the isoform with the highest expression level, and found that changes do not prevent conjugation to substrate proteins in vivo. Replacement of this conserved Gln by alanine resulted in a protein that was less readily removed from a substrate by SUMO protease EARLY IN SHORT DAYS4 in an in vitro reaction and apparently led to higher levels of SUMO conjugates when expressed in vivo. We used the SUMO1 variant with the Gln-to-alanine substitution, as well as SUMO3 and SUMO5 (which carry methionine and leucine, respectively, at this position), to enrich in vivo substrates. Identification of the most abundant proteins contained in these fractions indicated that they are involved in DNA-related, or in RNA-dependent, processes, such as regulation of chromatin structure, splicing, or translation. The majority of the identified bona fide substrates contain predicted sumoylation sites. A subset of the proteins was expressed in Escherichia coli and could be sumoylated in vitro.

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenic phenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuated vaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics system developed for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Vero or chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78, IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for the corresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast, viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classical virulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could not be recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains the determinants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulent strain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains the determinants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of the D78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues <em>Gln</em> at position 253 (<em>Gln</em>253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism, and pathogenic phenotype of virulent IBDV.

The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven intrachain disulfide bridges have been placed (Cys25-Cys63, Cys228-Cys276, Cys246-Cys264, Cys255-Cys408, Cys572-Cys748, Cys619-Cys666, Cys798-Cys826, Cys824-Cys860, Cys898-Cys1298, Cys1056-Cys1104, and Cys1329-Cys1444). Cys-447 probably forms an interchain bridge with Cys-447 from another subunit. The beta-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically regulated systems with particular reference to the complement components C3 and C4.

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

Single-amino-acid mutations in Sindbis virus proteins can convert clinically silent encephalitis into uniformly lethal disease. However, little is known about the host gene response during avirulent and virulent central nervous system (CNS) infections. To identify candidate host genes that modulate alphavirus neurovirulence, we utilized GeneChip Expression analysis to compare CNS gene expression in mice infected with two strains of Sindbis virus that differ by one amino acid in the E2 envelope glycoprotein. Infection with Sindbis virus, dsTE12H (E2-55 HIS), resulted in 100% mortality in 10-day-old mice, whereas no disease was observed in mice infected with dsTE12Q (E2-55 GLN). dsTE12H, compared with dsTE12Q, replicated to higher titers in mouse brain and induced more CNS apoptosis. Infection with the neurovirulent dsTE12H strain was associated with both a greater number of host genes with increased expression and greater changes in levels of host gene expression than was infection with the nonvirulent dsTE12Q strain. In particular, dsTE12H infection resulted in greater increases in the levels of mRNAs encoding chemokines, proteins involved in antigen presentation and protein degradation, complement proteins, interferon-regulated proteins, and mitochondrial proteins. At least some of these increases may be beneficial for the host, as evidenced by the demonstration that enforced expression of the antiapoptotic mitochondrial protein peripheral benzodiazepine receptor (PBR) protects neonatal mice against lethal Sindbis virus infection. Thus, our findings identify specific host genes that may play a role in the host protective or pathologic response to neurovirulent Sindbis virus infection.

The catalytic core domain (CCD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) harbors the enzyme active site and binds viral and chromosomal DNA during integration. Thirty-five CCD mutant viruses were constructed, paying particular attention to conserved residues in the Phe(139)-Gln(146) flexible loop and abutting Ser(147)-Val(165) amphipathic alpha helix that were implicated from previous in vitro work as important for DNA binding. Defective viruses were typed as class I mutants (specifically blocked at integration) or pleiotropic class II mutants (additional particle assembly and/or reverse transcription defects). Whereas HIV-1(P145A) and HIV-1(Q146K) grew like the wild type, HIV-1(N144K) and HIV-1(Q148L) were class I mutants, reinforcing previous results that Gln-148 is important for DNA binding and uncovering for the first time an important role for Asn-144 in integration. HIV-1(Q62K), HIV-1(H67E), HIV-1(N120K), and HIV-1(N155K) were also class I mutants, supporting findings that Gln-62 and Asn-120 interact with viral and target DNA, respectively, and suggesting similar integration-specific roles for His-67 and Asn-155. Although results from complementation analyses established that IN functions as a multimer, the interplay between active-site and CCD DNA binding functions was unknown. By using Vpr-IN complementation, we determined that the CCD protomer that catalyzes integration also preferentially binds to viral and target DNA. We additionally characterized E138K as an intramolecular suppressor of Gln-62 mutant virus and IN. The results of these analyses highlight conserved CCD residues that are important for HIV-1 replication and integration and define the relationship between DNA binding and catalysis that occurs during integration in vivo.

The role of the asparagine residue in the Cys-His-Asn "catalytic triad" of cysteine proteases has been investigated by replacing Asn175 in papain by alanine and glutamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters determined against the substrate carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)- L-arginine. At the optimal pH of 6.5, the specificity constant (k(cat)/KM)obs was reduced by factors of 3.4 and 150 for the Asn175->>Gln and Asn175->>Ala mutants, respectively. Most of this effect was the result of a decrease in k(cat), as neither mutation significantly affected KM. Substrate hydrolysis by these mutants is still much faster than the non-catalytic rate, and therefore Asn175 cannot be considered as an essential catalytic residue in the cysteine protease papain. Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn175 side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alteration of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the asparagine residue to the structural integrity of papain. The strict conservation of Asn175 in cysteine proteases might therefore result from a combination of functional and structural constraints.

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (GlnGlnGlnGlnGlnGlnGln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Substitution of Thr26 by <em>Gln</em> in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of <em>Gln</em>26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (<em>Gln</em>26 and His18, or <em>Gln</em>26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.

ATP-gated P2X(2) channels undergo activation-dependent permeability increases as they proceed from the selective I(1) state to the I(2) state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X(2) channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X(2) channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I(1) state to the dilated I(2) state. Initial channel opening to the I(1) state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I(2) state. Ten residues appeared to perturb only the I(1)-I(2) transition (Phe(31), Arg(34), Gln(37), Lys(53), Ile(328), Ile(332), Ser(340), Gly(342), Trp(350), Leu(352)). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X(2) channels.

N-terminal amino acid sequencing, ion spray mass spectrometry, and cleavage of synthetic peptide substrates were used to identify the N and C termini of the mature Gag and Pol proteins of feline immunodeficiency virus (FIV). The Gag polyprotein encodes matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. The Gag-Pol polyprotein encodes, in addition to the above proteins, protease (PR), reverse transcriptase (RT), dUTPase (DU), and integrase (IN). Secondary cleavage of RT at Trp-595-Tyr-596 of Pol yields a truncated form lacking the C-terminal RNase H domain. The observed and expected molecular masses of the viral proteins were in agreement, with three exceptions. (i) The molecular mass of MA was 14,735 Da, compared with a predicted mass of 14,649 Da, based on a single cleavage at Tyr-135-Pro-136 of Gag. The observed molecular mass is consistent with myristoylation of MA, which was confirmed by metabolic labeling of FIV MA with [3H]myristic acid. (ii) The N terminus of the NC protein is generated via cleavage at Gln-366-Val-367 of Gag, which predicts a mass of 25,523 for CA and 9,101 for the major form of NC. The observed mass of CA was 24,569, consistent with loss of nine C-terminal amino acids by a second cleavage of CA at Leu-357-Leu-358. Synthetic FIV protease accurately cleaved synthetic peptide substrates containing this site. (iii) The actual mass of NC (7,120 Da) was approximately 2 kDa smaller than the mass predicted by synthesis to the stop codon at the end of Gag (9,101 Da). Experiments are in progress to characterize additional cleavage(s) in NC.

BACKGROUND

The amino acid glutamine (GLN) has received considerable attention as a potential therapeutic adjuvant in critical illness and in improving postoperative clinical outcomes. Most studies on the role of GLN in cellular physiology have historically focused on its anabolic roles in specific cell types and its contribution to growth in cancer cells. However, an emerging body of work that examines the consequences of GLN deprivation on cellular survival and gene expression has constructed a new paradigm for this amino acid, namely, that limited extracellular GLN supplies modulate stress and apoptotic responses.

METHODS

A survey of the scientific literature was conducted on GLN in cell survival signaling and apoptosis. Work from our laboratory in liver cancer cells also was included in this review.

RESULTS

Most studies on this topic have used mammalian cell lines derived from the gut, immune system (including hybridomas), and various cancers. GLN limitation, even in the presence of an adequate glucose supply, impacts stress-related gene expression, differentially modulates receptor-mediated apoptosis, and directly elicits apoptosis through signaling mechanisms and caspase cascades that are specific to cell type. To date, GLN transporters, cellular hydration, glutaminyl-tRNA synthetase, ATP levels, mRNA stability, and glutathione economy have been variably implicated in GLN-dependent survival signaling.

CONCLUSIONS

The cell type-specific mechanisms underlying the regulatory role of GLN in cell survival continue to unfold at a steady pace through in vitro studies. These results have collectively provided testable hypotheses for further in vivo studies into their physiological relevance during GLN "nutritional pharmacology."

Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the epsilon-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels.

The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an alpha-helical pattern of exposed and buried positions spanning residues 270-309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinase-activating state. Each of the lock-on disulfides cross-links the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membrane-bound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.

The isolated N-terminal SH3 domain of the Drosophila adapter protein drk (drkN SH3 domain) exists in a dynamic equilibrium between a folded (F(exch)) and an unfolded (U(exch)) state under native-like buffer conditions [Zhang, O., & Forman-Kay, J. D. (1995) Biochemistry 34, 6784-6794]. The effect of binding a proline-rich peptide derived from the protein Sos, a biological target of the drkN SH3 domain, on this equilibrium has been investigated. The stabilization of the F(exch) folded state upon binding provides an example of the link between binding and protein folding or stabilization. We have compared NMR parameters of the U(exch) state with those of a denatured state in 2 M guanidine hydrochloride (U(Gdn)). Variable-temperature experiments demonstrate that interactions in a region encompassing residues Gln 23-Leu 28 in the U(exch) state are destabilized upon addition of guanidine hydrochloride. Data from an 15N HSQC-NOESY-HSQC experiment as well as recently developed methods provide more unambiguous structural information than described previously, showing the presence of preferential structure in both unfolded states. Backbone NOEs observed in both unfolded states as well as chemical shifts and coupling constants suggest a rapid equilibrium between extended structure and turn-like structures which may play a role in initiation of protein folding. However, differences in detailed structural features between the two unfolded states argue that caution is needed in interpretation of results from structural characterization of protein conformational states generated using denaturing conditions.

A synthetic 23-mer peptide that mimics the sequence of the putative transmembrane M2 segment of the Torpedo californica acetylcholine receptor (AcChoR) delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe-Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms discrete ionic channels in phosphatidylcholine bilayers. In contrast, a synthetic peptide that mimics the sequence of the putative M1 transmembrane segment of the Torpedo AcChoR delta subunit--Leu-Phe-Tyr-Val-Ile-Asn-Phe-Ile-Thr-Pro-Cys-Val-Leu-Ile-Ser-Phe- Leu-Ala-Ser-Leu-Ala-Phe-Tyr--does not form channels. The synthetic M2 delta channel peptide exhibits features that are characteristic of the authentic AcChoR channel, such as single channel conductances, discrimination of cations over anions, and channel lifetimes for open and closed states in the millisecond time range. Energetic considerations suggest that an aggregate of five amphipathic alpha-helices conforms the channel. Thus, the M2 segment may be a component of the AcChoR channel structure.

The X-ray repair cross-complementation group 1 (XRCC1) protein plays an important role in base excision repair. Several polymorphisms in the XRCC1 gene have been described, including Arg399Gln. Previous studies investigating the association between genetic polymorphism of Arg399Gln XRCC1 and risk of breast cancer have provided inconsistent results. A meta-analysis was conducted to investigate the association between common genetic variant in the XRCC1 gene (exon 10, Arg399Gln) with breast cancer risk. We identified 36 eligible studies, in relation to the Arg399Gln polymorphism of XRCC1 and risk of breast cancer. These studies comprised of 43,716 subjects (20,837 patients and 22,879 controls). We first estimated the risk of the genotypes Arg/Gln and Gln/Gln compared with the wild-type Arg/Arg homozygote, and then evaluated the risk of Gln/Gln versus (Arg/Gln+Arg/Arg) and (Gln/Gln+Arg/Gln) versus Arg/Arg, which assumed recessive and dominant effects, respectively, of the variant 399Gln allele. There was significant heterogeneity between studies. The overall ORs showed that the breast cancer risk were not associated with the XRCC1 genotypes. The heterogeneity between studies decreased dramatically when studies stratified into Asian and Western countries. There was significant association between the polymorphism of XRCC1 and breast cancer risk among studies of Asian countries. In Asian countries the Arg/Gln versus Arg/Arg (OR = 0.98, 95% CI: 0.88-1.10) and Gln/Gln+Arg/Gln versus Arg/Arg (OR = 1.05, 95% CI: 0.95-1.18) were not associated with increased risk of breast cancer. On the other hand, both Gln/Gln versus Arg/Arg (OR = 1.46, 95% CI: 1.19-1.79) and Gln/Gln versus Arg/Gln+Arg/Arg (OR = 1.49, 95% CI: 1.22-1.81) increased the risk. Therefore, it could be concluded that 399Gln allele might act as a recessive allele in its association with breast cancer risk.

Although hydrophobic interaction is the main contributing factor to the stability of the protein fold, the specificity of the folding process depends on many directional interactions. An analysis has been carried out on the geometry of interaction between planar moieties of ten side chains (Phe, Tyr, Trp, His, Arg, Pro, Asp, Glu, Asn and Gln), the aromatic residues and the sulfide planes (of Met and cystine), and the aromatic residues and the peptide planes within the protein tertiary structures available in the Protein Data Bank. The occurrence of hydrogen bonds and other nonconventional interactions such as C-H...pi, C-H...O, electrophile-nucleophile interactions involving the planar moieties has been elucidated. The specific nature of the interactions constraints many of the residue pairs to occur with a fixed sequence difference, maintaining a sequential order, when located in secondary structural elements, such as alpha-helices and beta-turns. The importance of many of these interactions (for example, aromatic residues interacting with Pro or cystine sulfur atom) is revealed by the higher degree of conservation observed for them in protein structures and binding regions. The planar residues are well represented in the active sites, and the geometry of their interactions does not deviate from the general distribution. The geometrical relationship between interacting residues provides valuable insights into the process of protein folding and would be useful for the design of protein molecules and modulation of their binding properties.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies (MAbs) against this protein can be arranged in groups, on the basis of a number of biological and biochemical properties. Group I antibodies are type-common, have high complement-independent neutralization titers, recognize discontinuous (conformational) epitopes, and block each other in a binding assay. The sum of their epitopes constitutes antigenic site I of gD. Using a panel of neutralization-resistant mutants, we previously found that group I MAbs can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies, and vice versa. Antigenic site I therefore consists of two parts, Ia and Ib. We have now identified the point mutations which prevent neutralization. Two Ib MAbs (DL11 and 4S) selected a Ser to Asn change at residue 140; this alteration creates a new N-linked glycosylation site, which is used. A third Ib MAb (D2) selected a Gln to Leu change at 132. The mutation selected by the Ia MAb HD1 (Ser to Asn at residue 216) is identical to that selected by MAb LP2, another Ia antibody. By using oligonucleotide-directed mutagenesis, we have produced gD genes with combinations of the above mutations. Attempts to recombine these genes into the virus genome were unsuccessful, suggesting that the combinations are lethal. This was confirmed by a complementation assay which measures the ability of gD transiently expressed in transfected Vero cells to rescue the production of infectious virus by the gD-minus mutant F-gD beta.

Glutamine (GLN) has been shown to protect against inflammatory injury and illness in experimental and clinical settings. The mechanism of this protection is unknown; however, laboratory and clinical trial data have indicated a relationship between GLN-mediated protection and enhanced heat shock protein 70 (HSP70) expression. The aim of this study was to examine the hypothesis that GLN's beneficial effect on survival, tissue injury, and inflammatory response after inflammatory injury is dependent on HSP70 expression. Mice with a specific deletion of the HSP70 gene underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 1 h post-CLP. Lung tissue NF-kappaB activation, inflammatory cytokine response, and lung injury were assessed post-CLP. Survival was assessed for 5 days post-CLP. Our results indicate that GLN administration improved survival in Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN; however, GLN exerted no survival benefit in Hsp70(-/-) mice. This was accompanied by a significant decrease in lung injury, attenuation of NF-kappaB activation, and proinflammatory cytokine expression in GLN-treated Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN. In the Hsp70(-/-) mice, GLN's attenuation of lung injury, NF-kappaB activation, and proinflammatory cytokine expression was lost. These results confirm our hypothesis that HSP70 expression is required for GLN's effects on survival, tissue injury, and the inflammatory response after global inflammatory injury.

Carbohydrates comprise about 50% of the mass of gp120, the external envelope glycoprotein of simian immunodeficiency virus (SIV) and human immunodeficiency virus. We identified 11 replication-competent derivatives of SIVmac239 lacking two, three, four, or five potential sites for N-linked glycosylation. These sites were located within and around variable regions 1 and 2 of the surface envelope protein of the virus. Asn (AAT) of the canonical N-linked glycosylation recognition sequence (Asn X Ser/Thr) was changed in each case to the structurally similar Gln (CAG or CAA) such that two nucleotide changes in the codon would be required for reversion. Replication of one triple mutant (g456), however, was severely impaired. A revertant of the g456 mutant was recovered from CEMx174 cells with a Met-to-Val compensatory substitution at position 144, 2 amino acids upstream of attachment site 5. Thus, a debilitating loss of sites for N-linked glycosylation can be compensated for by amino acid changes not involving the Asn-X-Ser/Thr consensus motif. These results provide a framework to begin testing the hypothesis that carbohydrates form a barrier that can limit the humoral immune responses to the virus.

The chemistry of peptide fragmentation by collision-induced dissociation (CID) is currently being reviewed, as a result of observations that the amino acid sequence of peptide fragments can change upon activation. This rearrangement mechanism is thought to be due to a head-to-tail cyclization reaction, where the N-terminal and C-terminal part of the fragment are fused into a macrocycle (= cyclic peptide) structure, thus "losing" the memory of the original sequence. We present a comprehensive study for a series of b fragment ions, from b(2) to b(8), based on the simplest amino acid residue glycine, to investigate the effect of peptide chain length on the appearance of macrocycle fragment structures. The CID product ions are structurally characterized with a range of gas-phase techniques, including isotope labeling, infrared photodissociation spectroscopy, gas-phase hydrogen/deuterium exchange (using CH(3)OD), and computational structure approaches. The combined insights from these results yield compelling evidence that smaller b(n) fragments (n = 2, 3) exclusively adopt oxazolone-type structures, whereas a mixture of oxazolone and macrocycle b fragment structures are formed for midsized b(n) fragments, where n = 4-7. As each of these chemical structures exchanges at different rates, it is possible to approximate the relative abundances using kinetic fits to the H/D exchange data. Under the conditions used here, the "slow"-exchanging macrocycle structure represents approximately 30% of the b ion population for b(6)-b(7), while the "fast"-exchanging oxazolone structure represents the remainder (70%). Intriguingly, for b(8) only the macrocycle structure is identified, which is also consistent with the "slow" kinetic rate in the HDX results. In a control experiment, a protonated cyclic peptide with 6 amino acid residues, cyclo(Gln-Trp-Phe-Gly-Leu-Met), is confirmed not to adopt an oxazolone structure, even upon collisional activation. These results demonstrate that in some cases larger macrocycle structures are surprisingly stable. While more studies are required to establish the general propensity for cyclization in b fragments, the implications from this study are troubling in terms of faulty sequence identification.