Administration of an elemental diet to rats given methotrexate (MTX), 20 mg/kg intraperitoneally (ip), results in 100% mortality from severe enterocolitis. Previous studies indicate that glutamine (GLN), which is not present in elemental diets, is the preferred oxidative substrate for the gut and may facilitate intestinal recovery after injury. This study investigated the effects of a glutamine-supplemented elemental diet (GLN-ED) on nutritional status, intestinal morphometry, bacterial translocation and survival in this lethal model of intestinal injury. Three experiments were performed. In the first experiment, rats received an intragastric elemental diet supplemented with either 2% GLN or an equivalent amount of glycine (Control). After 4 days animals received either MTX, 20 mg/kg ip, or saline ip and were killed 3 days later. The GLN-ED resulted in significantly decreased weight loss, improved nitrogen retention, and increased mucosal weight, protein, and DNA content of the jejunum and colon. In the second experiment rats were assigned to diet as in the first experiment, but all animals received MTX. Control diet animals died within 120 hrs of MTX administration. The GLN-ED group had significantly longer survival time and decreased mortality. In the third experiment animals were assigned to diet and MTX as in the first experiment. Ninety-six hrs later aortic blood cultures revealed enteric bacteremia in animals administered MTX. GLN-ED resulted in a significant reduction in the incidence of bacteremia. These experiments showed that a GLN-ED significantly improved nutritional status, decreased intestinal injury, decreased bacterial translocation, and resulted in improved survival in a lethal model of enterocolitis.

King et al. (King, K., Benkovic, S. J., and Modrich, P. (1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition. We have substituted Gln or Asp for Glu-111 by oligonucleotide-directed mutagenesis. First and second strand cleavage rate constants are reduced by a factor of more than 10(4) by the Gln-111 substitution. However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI sites by wild type endonuclease to a similar degree. The specific affinity of Gln-111 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation. In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-111 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme. These results suggest that a negative charge on residue 111 is required for strand cleavage and are consistent with participation of Glu-111 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.

Autoproteolysis of the retroviral aspartyl proteases is a major obstacle to purification and analysis of these enzymes. A mutagenic approach to rendering autolytic cleavage sites less labile was applied to the primary cleavage site between Leu5 and Trp6 in human immunodeficiency virus-1 (HIV-1) protease. From predictions based on known substrates it was concluded that amino acids Lys or Ser in place of Gln at position 7 would prevent cleavage at the Leu5-Trp6 peptide bond, therefore stabilizing the protein. Autoproteolytic stability was enhanced at least 100-fold by these mutations. At longer time points the protease was degraded at secondary sites which contained adequate substrate sequences but were conformationally restricted. Conversely, a mutation in HIV-2 protease which changed Lys7 to Gln rendered the protein 3-fold less stable and shifted the position of the initial autoproteolytic cleavage from Phe3-Ser4 to Leu5-Trp6. The effects of these mutations demonstrate that small changes in protein sequence can have a major impact on their autoproteolytic stability. The work described here suggests a general method for stabilizing proteases and perhaps other recombinantly produced proteins to autolysis.

The P2Y1 receptor is a membrane-bound G protein-coupled receptor stimulated by adenine nucleotides. Using alanine scanning mutagenesis, the role in receptor activation of charged amino acids (Asp, Glu, Lys, and Arg) and cysteines in the extracellular loops (EL) of the human P2Y1 receptor has been investigated. The mutant receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C induced by the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In addition to single point mutations, all receptors carried the hemagglutinin epitope at the N- terminus for detection of cell-surface expression. The C124A and C202A mutations, located near the exofacial end of transmembrane helix 3 and in EL2, respectively, ablated phospholipase C stimulation by </=100 microM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many other G protein-coupled receptors, is required for proper trafficking of the P2Y1 receptor to the cell surface. In contrast, the C42A and C296A mutant receptors (located in the N-terminal domain and EL3) were activated by 2-MeSADP, but the EC50 values were >1000-fold greater than for the wild-type receptor. The double mutant receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that Cys42 and Cys296 form another disulfide bridge in the extracellular region, which is critical for activation. Replacement of charged amino acids produced only minor changes in receptor activation, with two remarkable exceptions. The E209A mutant receptor (EL2) exhibited a >1000-fold shift in EC50. However, if Glu209 were substituted with amino acids capable of hydrogen bonding (Asp, <em>Gln</em>, or Arg), the mutant receptors responded like the wild-type receptor. Arg287 in EL3 was impaired similarly to Glu209 when substituted by alanine. Substitution of Arg287 by lysine, another positively charged residue, failed to fully restore wild-type activity.

We have previously shown that varying the N-terminal amino acid in alpha-helical peptides can cause large variations in helix content (Chakrabartty et al., 1993a). The Lifson-Roig theory for the helix-coil transition predicts, however, that substitutions at the N-terminus in an unacetylated peptide should have no effect on alpha-helix stability. We have therefore modified the theory to include these N-capping effects by assigning a statistical weight (the "n-value") to the amino acid immediately preceding a stretch of helical residues. The n-value measures the N-capping propensity of an amino acid, and like the helix propensity (w-value), it is independent of neighboring residues or positions in sequence. The new theory was used, with the experimental data for these substitutions, to calculate n-values and, hence, free energies for N-capping for the amino acids Gln, Ala, Val, Met, Pro, Ile, Leu, Thr, Gly, Ser, and Asn as well as for the acetyl group, which is commonly used to cap peptides. The free energies vary by approximately 1 kcal mol-1 from Gln (worst) to Asn (best), and the acetyl group is nearly as effective as Asn. N-Capping free energies were also found for Leu, Thr, Gly, Ser, and Asn when the N-terminus is charged at pH 5. The unfavorable effect of protonation of the N-terminus in an alpha-helix was found to be approximately 0.5 kcal mol-1. Our results agree well with a survey of N-capping preferences from protein crystal structures and are compared to results from site-directed mutagenesis of N-caps in proteins.

Glutaminyl-transfer RNA (Gln-tRNA(Gln)) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNA(Gln) by the heterodimeric Glu-tRNA(Gln) amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNA(Gln) at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNA(Gln) mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNA(Gln) recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.

The protective functions of saliva are attributed, in part, to its serous and mucous glycoproteins. We have studied, as representative molecules, the proline-rich glycoprotein (PRG) from human parotid saliva and the high (MG1) and low (MG2) molecular weight mucins from submandibular-sublingual saliva. PRG (38.9 kDa) contains 40% carbohydrate consisting of 6 triantennary N-linked units and a single peptide chain of 231 amino acids, 75% of which = PRO + GLY + GLN. PRG's secondary structure is comprised of 70% random coil (naked regions) and 30% beta-turns (glycosylated domains). MG1 (greater than 10(3) kDa) contains 15% protein (several disulfide linked subunits), 78% carbohydrate (290 units of 4-16 residues), 7% sulfate, and small amounts of covalently linked fatty acids. MG2 (200-250 kDa) contains 30% protein (single peptide chain), 68% carbohydrate (170 units of 2-7 residues), and 2% sulfate. The major carbohydrate units of MG2 are: NeuAc alpha 2,3Gal beta 1,3GalNAc,Gal beta 1,3GalNAc, and Fuc alpha 1,2Gal beta 1,3GalNAc. MG1 contains hydrophobic domains, as evidenced by its ability to bind fluorescent hydrophobic probes; MG2 does not. Collectively, the biochemical and biophysical comparisons between MG1 and MG2 indicate that these two mucins are structurally different. Several functional properties of MG1, MG2, and PRG have been examined, including their presence in two-hour in vivo enamel pellicle, binding to synthetic hydroxyapatite, lubricating properties, and interactions with oral streptococci. The data presented suggest that these glycoproteins may have multiple functions which are predicated, in part on their carbohydrate units. The potential significance of the structure-function relationships of these glycoproteins to the oral ecology is discussed.

The overexpression of the c-src gene product, pp60c-src, in avian and rodent embryo cells is not sufficient to induce cellular transformation. In this study we report that structural alterations within an amino terminal domain of pp60c-src, the exon 3 domain (residues 84-115) activate the oncogenic potential of the c-src gene product. Site-directed mutagenesis of the c-src gene was used to generate c-src variants encoding pp60c-src proteins with the following amino acid alterations: tyr 90 to phe (pm90F); tyr 92 to phe (pm92F); arg 95 to either trp, lys, glu or gln (pm95W, 95K, 95E and 95Q, respectively), and deletion of residues 92-95 (dl92). C-src variants encoding proteins with the alteration of arg 95 to trp, glu, or lys, or containing the deletion of residues 92-95, induced alterations in cell morphology and promoted growth in soft agar as well as changes in glucose transport and in vivo tyrosine phosphorylation of cellular proteins (including calpactin I heavy chain, p36). Analysis of in vivo phosphorylation of the transforming variant src proteins revealed little detectable alteration in the phosphorylation of tyr 527, a putative site of tyrosine kinase regulation. Our results suggest that structural alterations within a domain distal to the catalytic (kinase) domain activate pp60c-src kinase activity and, concomitantly, oncogenic potential. Furthermore, we suggest that the exon 3 domain of pp60c-src may contribute to the regulation and/or substrate specificity of the c-src protein.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.

Aberrant arachidonic acid metabolism by cyclooxygenase (COX)-2 and 12-lipoxygenase (LOX) has implicated in carcinogenesis. Genetic polymorphisms in COX-2 and 12-LOX might therefore affect susceptibility to colorectal cancer (CRC). To examine this hypothesis, genotypes of COX-2 -1290A>G, -1195G>A, -765G>C and 12-LOX 261Arg>Gln polymorphisms were determined in 1000 CRC patients and 1300 controls. Increased risk of developing CRC was associated with the COX-2 -1195GA [adjusted odds ratio (OR) = 1.24, 95% confidence interval (CI) = 1.00-1.54] and -1195AA (adjusted OR = 1.77, 95% CI = 1.38-2.25) genotypes compared with the -1195GG genotype. Similarly, the increased risk for CRC was also associated with the COX-2 -765GC genotype (adjusted OR = 1.73, 95% CI = 1.23-2.43) compared with the -765GG genotype. Consistent with the results of genotype analyses, the ORs for the A_(1195)-C_(765)-containing haplotypes were significantly higher than those for the G_(1195)-G_(765)-containing haplotypes (P < 0.01). Furthermore, the -1195A allele was further associated with advanced CRC, with adjusted ORs of Dukes D CRC against Dukes A CRC being 2.43 (95% CI = 1.15-4.97) and 2.66 (95% CI = 1.23-5.74) for the -1195GA and -1195AA genotypes, respectively. The increased risk of CRC was also associated with the 12-LOX 261Gln/Gln genotype compared with the Arg/Arg genotype (adjusted OR = 1.38, 95% CI = 1.09-1.74). Together, these observations indicate that inherited polymorphisms in arachidonic acid-metabolizing enzymes may confer susceptibility to CRC.

An apolipoprotein, designated by its carboxyl-terminal residue as apoLp-Gln-II, has been isolated from the human high-density lipoprotein family, and its complete aminoacid sequence was determined. The apoprotein, one of the two major apoproteins of this family, is composed of two identical polypeptide chains, each containing 77 amino acids. The two polypeptide chains are connected by a single disulfide bridge at position 6 in the sequence. The minimum molecular weight of the intact apoprotein is 17,380. The amino-terminal residue of each chain is pyrrolidone carboxylic acid, and the carboxyl-terminal residue is glutamine.

The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a free-energy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochrome c heme edge is within 4 A of the indole ring of subunit II residue Trp(104), indicating a likely electron transfer path. These two groups are surrounded by a small, hydrophobic contact region, which is surrounded by electrostatically complementary hydrophilic interactions. Cytochrome c/cytochrome c oxidase interactions of Lys(13) with Asp(119) and Lys(72) with Gln(103) and Asp(158) are the most critical polar interactions due to their proximity to the hydrophobic region and exclusion from bulk solvent. The predicted complex matches previous mutagenesis, binding, and time-resolved kinetics studies that implicate Trp(104) in electron transfer and show the importance of specific charged residues to protein affinity. Electrostatic forces not only enhance long range protein/protein association; they also predominate in short range alignment, creating the transient interaction needed for rapid turnover.

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region-like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region-like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.

Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin-binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408->>Glu/Lys939->>Arg mutant retained significant activity, while an Asp407->>Glu/Lys939->>Arg mutant exhibited only marginal function. An Asp407->>Glu/Asp408->>Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407->>Glu/Asp408->>Glu/Lys939->>Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K).

Urotensin I, purified from extracts of the urophysis of a teleost fish (Catostomus commersoni), exhibits potent hypotensive activity (mammals and birds) and corticotropin-releasing activity (both fish and mammals). The primary structure of this 41-residue peptide was determined to be H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Asn-Met-Ile-Glu- Met-Ala-Arg-Ile-Glu-Asn-Glu-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu -Val-NH2. Extraction with 0.1N HCl at 100 degrees C cleaves the amino-terminal tripeptide, yeilding a fully active analog, urotensin I(4-41). The amino acid sequence was confirmed by measuring the biological activity of synthetic urotensin I(4-41). Urotensin I exhibits a striking sequence homology with ovine corticotropin-releasing factor and with frog sauvagine. These three peptides exhibit similar activities in biological test systems.

Excision repair cross-complementing group 2 (ERCC2), a major DNA repair protein, is involved in nucleotide excision repair and basal transcription. The ERCC2 polymorphisms have been associated with altered DNA repair capacity. We investigated two ERCC2 polymorphisms, Asp312Asn and Lys751Gln, in 1092 Caucasian lung cancer patients and 1240 spouse and friend controls. The results were analyzed using generalized additive models and logistic regression, adjusting for relevant covariates. The overall adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were 1.47 (95% CI, 1.1-2.0) for the Asp312Asn polymorphism (Asn/Asn versus Asp/Asp) and 1.06 (95% CI, 0.8-1.4) for the Lys751Gln polymorphism (Gln/Gln versus Lys/Lys). Gene-smoking interaction analyses revealed that the adjusted ORs for each of the two polymorphisms decreased significantly as pack-years increased. When comparing individuals with Asn/Asn + Gln/Gln versus individuals with Asp/Asp + Lys/Lys, the fitted ORs (95% CIs) were 2.56 (95% CI, 1.3-5.0) in nonsmokers and 0.69 (95% CI, 0.4-1.2) in heavy smokers (80 pack-years; P < 0.01 for the interaction term). Consistent and robust results were found when models incorporated different definitions of cumulative cigarette smoking. A stronger gene-smoking interaction was observed for the Asp312Asn polymorphism than for the Lys751Gln polymorphism. In conclusion, cumulative cigarette smoking modifies the associations between ERCC2 polymorphisms and lung cancer risk.

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled-coil protein in determining coiled-coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three-stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys-Gly-Gly linker, unfolding occurred from the same two-stranded monomeric oligomerization state for all of the analogs. Denaturation from a two-stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled-coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed-phase high-performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled-coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high-performance size-exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 microM under benign buffer conditions. The Leu-, Tyr-, Gln-, and His-substituted analogs were found to be exclusively three-stranded trimers, while the Asn-, Lys-, Orn-, Arg-, and Trp-substituted analogs formed exclusively two-stranded monomers. Modeling results for the Leu-substituted analog showed that a three-stranded oligomerization state is preferred due to increased side-chain burial, while a two-stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three-stranded state.

Molecular mimicry or epitope similarity between group A streptococcal M proteins and myosin may contribute to the presence of heart reactive antibodies in acute rheumatic fever. In our study overlapping synthetic peptides copying the entire sequence of PepM5 protein were used to map the myosin cross-reactive epitopes of streptococcal M protein recognized by mouse and human mAb and affinity purified myosin-specific antibodies from acute rheumatic fever and rheumatic heart disease sera. Overlapping M protein peptides SM5(164-197)C and SM5(184-197)C inhibited the murine mAb reactions with PepM5 protein. The human mAb and affinity purified myosin-specific antibodies reacted exclusively with SM5(184-197)C. However, one of the five different purified myosin-specific antibodies not only reacted with SM5(184-197)C but also reacted with SM5(84-116)C. The synthetic subpeptides SM5(175-184)C and SM5(188-197C) did not react with any of the antibodies to PepM5 and myosin demonstrating a requirement of the 184-188 amino acid sequence for antibody recognition. A heptapeptide containing the sequence SM5(183-189) was also found to inhibit selected human myosin-specific antibodies and a human antimyosin mAb. Therefore, the majority of mouse and human myosin crossreactive antibodies recognized an epitope within the 14 residue carboxy terminus of PepM5 which appeared to involve the GLN-LYS-SER-LYS-GLN sequence.

Branched-chain ketoacid dehydrogenase deficiency results in complex and volatile metabolic derangements that threaten brain development. Treatment for classical maple syrup urine disease (MSUD) should address this underlying physiology while also protecting children from nutrient deficiencies. Based on a 20-year experience managing 79 patients, we designed a study formula to (1) optimize transport of seven amino acids (Tyr, Trp, His, Met, Thr, Gln, Phe) that compete with branched-chain amino acids (BCAAs) for entry into the brain via a common transporter (LAT1), (2) compensate for episodic depletions of glutamine, glutamate, and alanine caused by reverse transamination, and (3) correct deficiencies of omega-3 essential fatty acids, zinc, and selenium widespread among MSUD patients. The formula was enriched with LAT1 amino acid substrates, glutamine, alanine, zinc, selenium, and alpha-linolenic acid (18:3n-3). Fifteen Old Order Mennonite children were started on study formula between birth and 34 months of age and seen at least monthly in the office. Amino acid levels were checked once weekly and more often during illnesses. All children grew and developed normally over a period of 14-33 months. Energy demand, leucine tolerance, and protein accretion were tightly linked during periods of normal growth. Rapid shifts to net protein degradation occurred during illnesses. At baseline, most LAT1 substrates varied inversely with plasma leucine, and their calculated rates of brain uptake were 20-68% below normal. Treatment with study formula increased plasma concentrations of LAT1 substrates and normalized their calculated uptakes into the nervous system. Red cell membrane omega-3 polyunsaturated fatty acids and serum zinc and selenium levels increased on study formula. However, selenium and docosahexaenoic acid (22:6n-3) levels remained below normal. During the study period, hospitalizations decreased from 0.35 to 0.14 per patient per year. There were 28 hospitalizations managed with MSUD hyperalimentation solution; 86% were precipitated by common infections, especially vomiting and gastroenteritis. The large majority of catabolic illnesses were managed successfully at home using 'sick-day' formula and frequent amino acid monitoring. We conclude that the study formula is safe and effective for the treatment of classical MSUD. In principle, dietary enrichment protects the brain against deficiency of amino acids used for protein accretion, neurotransmitter synthesis, and methyl group transfer. Although the pathophysiology of MSUD can be addressed through rational formula design, this does not replace the need for vigilant clinical monitoring, frequent measurement of the complete amino acid profile, and ongoing dietary adjustments that match nutritional intake to the metabolic demands of growth and illness.

The complete form of androgen insensitivity is an inherited X-linked syndrome in which genetic males fail to undergo masculinization in utero due to defective functioning of the androgen receptor (AR). The molecular basis of androgen insensitivity was investigated in the testicular feminized (Tfm) rat with this syndrome. AR mRNA size and amount, as well as nuclear AR protein revealed by immunocytochemistry, suggested normal expression of the AR gene in the Tfm rat. Sequence analysis of the AR coding region from Tfm and wild-type littermate male rats revealed a single transition mutation, guanine to adenine, within exon E, changing arginine 734 to glutamine within the steroid-binding domain of the AR. This arginine is highly conserved among the family of nuclear receptors and may be part of a phosphorylation recognition site. A recreated mutant AR (Arg734----Gln) expressed in COS cells had only 10-15% of the androgen-binding capacity of wild-type AR; the reduced androgen-binding capacity was similar to that of AR in tissue extracts of the Tfm rat. Stimulation of transcriptional activity by the recreated mutant AR was reduced relative to wild-type AR in cotransfection assays in CV1 cells using as reporter plasmid the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. Thus, arginine 734 appears essential for normal AR function both in androgen binding and transcriptional activation. Absence of these functions results in androgen insensitivity and lack of male sexual development.

EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln(12) and Glu(14) and ephrin-B2. Mutation of Gln(12) and Glu(14) does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has approximately 10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln(12) and Glu(14) with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes.