p53-mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem (ES) cells that express p53 with <em>Gln</em> and Ser in place of Leu25 and Trp26, respectively. p53(<em>Gln</em>25Ser26) was stable but did not accumulate after DNA damage; the expression of p21/Waf1 and PERP was not induced, and p53-dependent repression of MAP4 expression was abolished. Therefore, p53(<em>Gln</em>25Ser26) is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53(<em>Gln</em>25Ser26) was phosphorylated at Ser18 but was not acetylated at C-terminal sites, and its DNA binding activity did not increase, further supporting a role for p53 acetylation in the activation of sequence-specific DNA binding activity. Most importantly, p53(<em>Gln</em>25Ser26) mouse thymocytes and ES cells, like p53(-/-) cells, did not undergo DNA damage-induced apoptosis. We conclude that the transcriptional activities of p53 are required for p53-dependent apoptosis.

cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15 degrees -98 degrees C) were used to generate 1111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gln, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60 degrees C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.

Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectable products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by gamma-rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys>> Met>> Trp>> Tyr>> Phe>> cystine>> His>> Leu, Ile>> Arg, Lys, Val>> Ser, Thr, Pro>> Gln, Glu>> Asp, Asn>> Ala>> Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectable. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise approximately 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

We recently found that scatter factor (SF), a cell motility factor with a multimodular structure, is identical to hepatocyte growth factor (HGF), a potent mitogen of various cell types. SF/HGF is the ligand of the c-Met receptor tyrosine kinase. Here we used transient expression of naturally occurring and in vitro mutagenized cDNAs of SF/HGF to delineate the protein domains necessary for biological activity and binding to the c-Met receptor. (i) A single-chain SF/HGF resulting from the destruction of the protease cleavage site between heavy and light chain (Arg-494->> Gln) was largely inactive, indicating that proteolytic cleavage is essential for acquisition of the biologically active conformation. (ii) A SF/HGF splice variant encoding a protein with a 5-amino acid deletion in the first kringle domain was as highly active as the wild-type molecule. (iii) The separately expressed light chain (with serine protease homology) was inactive in all assays tested. (iv) The separate heavy chain as well as a naturally occurring splice variant consisting of the N terminus and the first two kringle domains bound the c-Met receptor, stimulated tyrosine auto-phosphorylation, and induced scattering of epithelial cells but not mitogenesis. These data indicate that a functional domain in the N terminus/first two kringle regions of SF/HGF is sufficient for binding to the Met receptor and that this leads to the activation of the downstream signal cascade involved in the motility response. However, the complete SF/HGF protein seems to be required for mitogenic activity.

ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.

A novel peptide called ghrelin or motilin-related-peptide (MTLRP) was found in the stomach of various mammals. We studied its effect on the motor function of the rat gastrointestinal tract. In normal, conscious unoperated animals, ghrelin/MTLRP (5 or 20 microg/kg iv) significantly accelerated the gastric emptying of a methylcellulose liquid solution (gastric residue after 15 min: 57 +/- 7, 42 +/- 11, 17 +/- 4, and 9 +/- 3% of the ingested meal with doses of 0, 1, 5, and 20 microg/kg iv, respectively) Transit of the methylcellulose liquid solution was also accelerated by ghrelin/MTLRP in the small intestine but not in the colon. Des-[Gln(14)]ghrelin, also found in the mammalian stomach, was as potent as ghrelin in emptying the stomach (gastric residue after 15 min: 12 +/- 3% at a dose of 20 microg/kg iv). In rats in which postoperative gastrointestinal ileus had been experimentally induced, ghrelin/MTLRP (20 microg/kg iv) reversed the delayed gastric evacuation (gastric residue after 15 min: 28 +/- 7% of the ingested meal vs. 82 +/- 9% with saline). In comparison, the gastric ileus was not modified by high doses of motilin (77 +/- 7%) or erythromycin (82 +/- 6%) and was only partially improved by calcitonin gene-related peptide (CGRP) 8-37 antagonist (59 +/- 7%). Ghrelin/MTLRP, therefore, accelerates the gastric emptying and small intestinal transit of a liquid meal and is a strong prokinetic agent capable of reversing the postoperative gastric ileus in rat.

The development of epitope-based vaccines, which have wide population coverage, is greatly complicated by MHC polymorphism. The grouping of alleles into supertypes, on the basis of common structural and functional features, addresses this problem directly. In the present study we applied a combined bioinformatics approach, based on analysis of both protein sequence and structure, to identify similarities in the peptide binding sites of 2225 human class II MHC molecules, and thus define supertypes and supertype fingerprints. Two chemometric techniques were used: hierarchical clustering using three-dimensional Comparative Similarity Indices Analysis fields and nonhierarchical k-means clustering using sequence-based z-descriptors. An average consensus of 84% was achieved, i.e., 1872 of 2225 class II molecules were classified in the same supertype by both techniques. Twelve class II supertypes were defined: five DRs, three DQs, and four DPs. The HLA class II supertypes and their fingerprints given in parenthesis are DR1 (Trp(9beta)), DR3 (Glu(9beta), Gln(70beta), and Gln/Arg(74beta)), DR4 (Glu(9beta), Gln/Arg(70beta), and Glu/Ala(74beta)), DR5 (Glu(9beta), Asp(70beta)), and DR9 (Lys/Gln(9beta)); DQ1 (Ala/Gly(86beta)), DQ2 (Glu(86beta), Lys(71beta)), and DQ3 (Glu(86beta), Thr/Asp(71beta)); DPw1 (Asp(84beta) and Lys(69beta)), DPw2 (Gly/Val(84beta) and Glu(69beta)), DPw4 (Gly/Val(84beta) and Lys(69beta)), and DPw6 (Asp(84beta) and Glu(69beta)). Apart from the good agreement between known binding motifs and our classification, several new supertypes, and corresponding thematic binding motifs, were also defined.

We recently cloned a full-length DNA copy of the dengue type 4 virus genome. Analysis of the 5' terminal nucleotide sequence suggested that the three-virion structural proteins are synthesized by proteolytic cleavage of a polyprotein precursor which is encoded in one open reading frame. We now present the remaining sequence of the dengue type 4 virus genome which codes for the nonstructural proteins. The entire genome, which is 10,644 nucleotides in length, contains one long open reading frame which codes for a single large polyprotein 3386 amino acids in length. Alignment of the dengue nonstructural protein sequence with that of other flaviviruses, including yellow fever and West Nile viruses, revealed that significant homology exists throughout the entire nonstructural region of the dengue genome and this allowed tentative assignment of individual nonstructural proteins in the following order: NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5-COOH. Processing of the nonstructural proteins appears to involve two types of proteolytic cleavage: the first occurs after a long hydrophobic signal sequence and the second occurs at a junction between two basic amino acids and a small polar amino acid. A notable exception is the cleavage at the N-terminus of the dengue NS3 which may take place at the junction between Gln-Arg and Ser. Comparative analysis suggests that dengue NS3 and NS5 may be involved in enzymatic activities related to viral replication and/or transcription. Putative nonstructural proteins NS2a, NS2b, NS4a, and NS4b are extremely hydrophobic, suggesting that these proteins are most likely associated with cellular membranes.

A point mutation in the heavy chain of cardiac myosin, resulting in replacement of an arginine (Arg) with glutamine (Gln), has been linked to hypertrophic cardiomyopathy in humans (Geisterfer-Lowrance, A. A. T., Kass, S., Tanigawa, G., Vosberg, H.-P., McKenna, W., Seidman, J. G., and Seidman, C. E. (1990) Cell 62, 999-1006). To determine the functional impact of this mutation, baculovirus-driven coexpression of myosin heavy and light chains has been developed. The Arg-403->>Gln mutation resulted in cardiac myosin with normal ATPase activity in the absence of actin. However, in the presence of actin, ATPase activity was greatly reduced (Vmax decreased>> 3.5-fold and K(app) increased>> 3-fold). In vitro motility was reduced nearly 5-fold by this single amino acid mutation. Thus, Arg-403 likely contributes to an important interaction at the actin interface of myosin. Replacement of Arg-403 with Gln leads to decreased rate(s) of transition within the actin-myosin crossbridge cycle. In humans, this mutation will result in decreased power output per unit area of cardiac muscle, likely providing a stimulus for hypertrophy.

Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64----Gly substitution produced a sterically unhindered myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Gly64 (approximately 10(7) M-1 s-1) much greater than Val64 approximately Leu64 (approximately 10(6) M-1 s-1) greater than Met64 greater than Phe64 approximately His64 approximately GlnGln approached or were larger than those for chelated model heme (i.e. approximately 1 x 10(8) M-1 s-1 for O2 and approximately 1 x 10(7) M-1 s-1 for CO), whereas the corresponding rate parameters for myoglobin containing either GlnGln, His64----Val, and His64----Leu mutants were compared to those reported previously for Asian elephant myoglobin (Gln-E7), Aplysia limacina myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).

Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX. If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27.

The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.

The aim of the present study was to assess the contribution of polymorphisms in the breast cancer resistance protein/ATP-binding cassette transporter G2 (BCRP/ABCG2) gene to the placental expression from a new perspective, allelic imbalance. Polymorphisms were screened by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis followed by sequencing with DNA extracted from 100 placentas. To examine whether polymorphisms of the BCRP gene correlate with the placental BCRP expression, we determined mRNA and protein levels by quantitative real-time PCR and Western blotting, respectively. In placentas, G34A (Val(12)Met) and C421A (Gln(141)Lys) were frequently observed (18-36%), but C376T, which creates a stop codon (Gln(126) stop codon), was found with an allelic frequency of 1%. The mean of the BCRP protein level was significantly lower (p < 0.05) in homozygotes for the A421 allele than in those for the C421 allele, and heterozygotes had an intermediate value. To evaluate whether the C421A polymorphism acts as a cis-element in BCRP transcription, allelic imbalance was determined using informative lymphoblasts and 56 samples of placental cDNA. In most of the placental samples we tested, the difference in expression levels between the two alleles was small, and only two samples indicated a monoallelic expression (i.e., preferential expression of one allele). These results suggest that 1) the predominant allelic expression pattern of BCRP in placental samples is biallelic, and 2) the mutation C421A is not a genetic variant acting in cis, but is considered to influence the translation efficiency.

Evidence that many protein regions and even entire proteins lacking stable tertiary and/or secondary structure in solution (i.e., intrinsically disordered proteins) might be involved in protein-protein interactions, regulation, recognition, and signal transduction is rapidly accumulating. These signaling proteins play a crucial role in the development of several pathological conditions, including cancer. To test a hypothesis that intrinsic disorder is also abundant in cardiovascular disease (CVD), a data set of 487 CVD-related proteins was extracted from SWISS-PROT. CVD-related proteins are depleted in major order-promoting residues (Trp, Phe, Tyr, Ile, and Val) and enriched in some disorder-promoting residues (Arg, Gln, Ser, Pro, and Glu). The application of a neural network predictor of natural disordered regions (PONDR VL-XT) together with cumulative distribution function (CDF) analysis, charge-hydropathy plot (CH plot) analysis, and alpha-helical molecular recognition feature (alpha-MoRF) indicator revealed that CVD-related proteins are enriched in intrinsic disorder. In fact, the percentage of proteins with 30 or more consecutive residues predicted by PONDR VL-XT to be disordered was 57 +/- 4% for CVD-associated proteins. This value is close that described earlier for signaling proteins (66 +/- 6%) and is significantly larger than the content of intrinsic disorder in eukaryotic proteins from SWISS-PROT (47 +/- 4%) and in nonhomologous protein segments with a well-defined three-dimensional structure (13 +/- 4%). Furthermore, CDF and CH-plot analyses revealed that 120 and 36 CVD-related proteins, respectively, are wholly disordered. This high level of intrinsic disorder could be important for the function of CVD-related proteins and for the control and regulation of processes associated with cardiovascular disease. In agreement with this hypothesis, 198 alpha-MoRFs were predicted in 101 proteins from the CVD data set. A comparison of disorder predictions with the experimental structural and functional data for a subset of the CVD-associated proteins indicated good agreement between predictions and observations. Thus, our data suggest that intrinsically disordered proteins might play key roles in cardiovascular disease.

Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.

BACKGROUND

Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with Kirsten-Ras (K-Ras) mutation status. Current mandatory mutation testing for patient selection is limited to the K-Ras 'hotspot' codons 12 and 13.

METHODS

Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis. Taqman-based gene copy number and FISH analyses were used to investigate K-Ras gene amplification.

RESULTS

Four additional K-Ras mutations (Leu(19)Phe (1 out of 106 tumours), Lys(117)Asn (1 out of 106), Ala(146)Thr (7 out of 106) and Arg(164)Gln (1 out of 106)) were identified. Lys(117)Asn and Ala(146)Thr had phenotypes similar to the hotspot mutations, whereas Leu(19)Phe had an attenuated phenotype and the Arg(164)Gln mutation was phenotypically equivalent to wt K-Ras. We additionally identified a new K-Ras gene amplification event, present in approximately 2% of tumours.

CONCLUSIONS

The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third. Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

Several potential functional polymorphisms (Arg194Trp, Arg280His, Arg399Gln) in the DNA base excision repair gene X-ray repair cross-complementing group 1 (XRCC1) have been implicated in cancer risk. Our meta-analysis on total of 11,957 cancer cases and 14,174 control subjects from 38 published case-control studies showed that the odds ratio (OR) for the variant genotypes (Trp/Trp + Arg/Trp) of the Arg194Trp polymorphism, compared with the wild-type homozygote (Arg/Arg), was 0.89 [95% confidence interval (95% CI), 0.81-0.98] for all tumor types without between-study heterogeneity. Similarly, the overall risk for the combined variant genotypes (His/His + Arg/His) of the Arg280His, compared with the wild homozygote (Arg/Arg), was 1.19 (95% CI, 1.00-1.42). However, there was no main effect in either recessive or dominant modeling for the Arg399Gln, and the variant Gln/Gln homozygote was not associated with overall cancer risk (OR, 1.01; 95% CI, 0.90-1.14). The analyses suggest that XRCC1 Arg194Trp, Arg280His polymorphisms may be biomarkers of cancer susceptibility and a single larger study with thousands of subjects and tissue-specific biochemical and biological characterization is warranted to further evaluate potential gene-to-gene and gene-to-environment interactions on XRCC1 polymorphisms and cancer risk.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5->>Gly, Tyr10->>Phe, and His13->>Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22->>Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3->>Arg, Arg5->>Glu, Asp7->>Val, His13->>Lys, Lys16->>His, Val18->>Asp, Phe19->>Ser, Phe20->>Tyr, Ser26->>Pro, Ala30->>Val, Ile31->>Ala, Met35->>norLeu, Gly38->>Ile, Val39->>Ala, and Val40->>Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.

Using site-directed mutagenesis we have created an altered calmodulin in which Gln-3 and Thr-146 have both been replaced by cysteines. We have reacted this protein with the bifunctional reagent, bismaleimidohexane, forming an intramolecular cross-link between the two cysteines. In the crystal structure of native calmodulin alpha-carbons at positions 3 and 146 are 37 A apart. In the bismaleimidohexane cross-linked protein these atoms can be no more than 19 A apart, and model building studies indicate that there is probably a bend in the central helix of calmodulin. A second modified calmodulin was generated by cleaving the central helix of the cross-linked protein at Lys-77 with trypsin. In this molecule, the two lobes of calmodulin are joined solely by the bismaleimidohexane cross-link, which bridges Cys-3 and Cys-146. Vm and Kact values for activation of myosin light chain kinase activity by the cross-linked and cross-linked/trypsinized proteins are not significantly different from those for the control protein. This result indicates that one role for the central helix may be to serve as a flexible tether between the calmodulin lobes. This is consistent with a model calmodulin-enzyme complex in which the central helix is bent, and the two lobes exert a concerted effect. A detailed model of this type has been proposed for the calmodulin-myosin light chain kinase complex (Persechini, A. and Kretsinger, R.H. (1988) J. Cardiovasc. Pharmacol., in press).

Excellent results have been obtained for the Fmoc solid-phase syntheses of peptides using the activating reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU). Activation occurs very rapidly in N,N-dimethylformamide and N-methyl-pyrrolidone, optimal solvents for peptide-resin solvation. It has been observed that complete coupling reactions occur in only 10-30 min. Residues such as Arg, Ile, Leu and Val, which often require double coupling by other activation methods, react with high efficiency by single coupling when HBTU is used. The Fmoc/HBTU chemistry has recently been applied to the peptide synthesizers. The incorporation of trityl side-chain protection for Fmoc-Asn and Fmoc-Gln further enhances coupling efficiencies in difficult sequences.

Proton magnetic resonance spectroscopy ((1)HMRS) is an in vivo brain imaging method that can be used to investigate psychotropic drug mechanism of action. This study evaluated baseline (1)HMRS spectra of bipolar depressed patients and whether the level of cerebral metabolites changed after an open trial of lamotrigine, an anti-glutamatergic mood stabilizer. Twenty-three bipolar depressed and 12 control subjects underwent a MRS scan of the anterior cingulate/medial prefrontal cortex. The scan was performed on a GE whole-body 1.5 T MRI scanner using single-voxel PRESS (TE/TR=30/3000 ms, 3 x 3 x 3 cm(3) and post-processed offline with LCModel. Baseline CSF-corrected absolute concentrations of glutamate+glutamine ([Glx]), glutamate ([Glu]), and creatine+phosphocreatine ([Cr]) were significantly higher in bipolar depressed subjects vs healthy controls. The non-melancholic subtype had significantly higher baseline [Glx] and [Glu] levels than the melancholic subtype. Remission with lamotrigine was associated with significantly lower post-treatment glutamine ([Gln]) in comparison to non-remission. These data suggest that non-melancholic bipolar depression is characterized by increased glutamate coupled with increased energy expenditure. Lamotrigine appears to reduce glutamine levels associated with treatment remission. Further study is encouraged to determine if these MR spectroscopic markers can delineate drug mechanism of action and subsequent treatment response.