To understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP). Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic level. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein. Glu46, replaced by Gln, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state. The Glu46Gln mutation shifted the ground state absorption maximum from 446 to 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds. Arg52, which separates the active site from solvent in the ground state, was substituted by Ala. The smaller red shift (to 452 nm) of the Arg52Ala mutant suggests that electrostatic interactions with Arg52 are not important for charge stabilization on the chromophore. Both mutations cause interesting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu46Gln PYP in response to a change in pH from pH 5 to 10 (pKa = 8). Prompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle. The rate of color loss decreased about 3-fold with increasing pH from pH 5 to 10. The rate of recovery to the colored ground state showed a bell-shaped pH dependence, controlled by two pKa values (6.4 and 9.4). The maximum recovery rate at pH 7.9 is about 16 times faster than at pH 5. The effect of pH on Arg52Ala is like that on wild type except for faster loss of color and slower recovery. These kinetic effects of the mutations and the changes with pH demonstrate that both phases in PYP's light cycle are actively controlled by the protein component.

In eukaryotes, a single translational release factor, eRF1, deciphers three stop codons, although its decoding mechanism remains puzzling. In the ciliate Tetrahymena thermophila, UAA and UAG codons are reassigned to Gln codons. A yeast eRF1-domain swap containing Tetrahymena domain 1 responded only to UGA in vitro and failed to complement a defect in yeast eRF1 in vivo at 37 degrees C. This finding demonstrates that decoding specificity of eRF1 from variant code organisms resides at domain 1. However, the wild-type eRF1 hybrid fully restored the growth of eRF1-deficient yeast at 30 degrees C. Tetrahymena eRF1 contains a variant sequence, KATNIKD, at the tip of domain 1. The TASNIKD variant of hybrid eRF1 rendered the eRF1-nullified yeast viable, although in an in vitro assay, the same hybrid eRF1 responded only to UGA. Nevertheless, the yeast eRF1 bearing the KATNIKD motif instead of the TASNIKS heptapeptide present in higher eukaryotes remains omnipotent in vivo. Collectively, these data suggest that variant genetic code organisms like Tetrahymena have an intrinsic potential to decode three stop codons in vivo, and that interaction within domain 1 between the KAT tripeptide and other sequences modulates the decoding specificity of Tetrahymena eRF1.

smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.

The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.

We have identified a novel gene transcript of approximately 1.1 kilobases in length that is expressed in the presumptive nasal epithelium of the mouse embryo. In situ hybridization analysis shows discrete regions of expression associated with the palate, nasal septum, and nasal conchae. This transcript is also expressed strongly in the trachea and bronchi of the adult lung. Screening of a mouse heart cDNA library yielded several overlapping clones to give a continuous sequence of 1113 bases, containing an open reading frame of 278 codons comprising the complete mRNA. No significant homologies with known genes were observed at the nucleotide level; limited amino acid homology with two salivary gland-specific proteins was noted. A search for functionally significant protein motifs revealed consensus sequences for N-glycosylation, protein kinase C and casein kinase phosphorylation, and a leucine zipper. Additionally, we observed a unique amino acid sequence pattern, consisting of the residues Gly-(Leu/Pro/Gln)-(Pro/Leu)-Leu-Pro-Leu, repeated four times near the amino-terminal portion of the protein with two amino acid residues separating the repeats. Based on these observations, we propose that we have identified a new gene, which we call plunc (for palate, lung, and nasal epithelium clone; GenBankTM accession number U69172).

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.

Ionizable residues play essential roles in proteins, modulating protein stability, fold and function. Asp, Glu, Arg, and Lys make up about a quarter of the residues in an average protein. Multi-conformation continuum electrostatic (MCCE) calculations were used to predict the ionization states of all acidic and basic residues in 490 proteins. Of all 36,192 ionizable residues, 93.5% were predicted to be ionized. Thirty-five percent have lost 4.08 kcal/mol solvation energy (DeltaDeltaG(rxn)) sufficient to shift a pK(a) by three pH units in the absence of other interactions and 17% have DeltaDeltaG(rxn) sufficient to shift pK(a) by five pH units. Overall 85% of these buried residues (DeltaDeltaG(rxn)>5DeltapK units) are ionized, including 92% of the Arg, 86% of the Asp, 77% of the Glu, and 75% of the Lys. Ion-pair interactions stabilize the ionization of both acids and bases. The backbone dipoles stabilize anions more than cations. The interactions with polar side-chains are also different for acids and bases. Asn and Gln stabilize all charges, Ser and Thr stabilize only acids while Tyr rarely stabilize Lys. Thus, hydroxyls are better hydrogen bond donors than acceptors. Buried ionized residues are more likely to be conserved than those on the surface. There are 3.95 residues buried per 100 residues in an average protein.

Limited subtilisin digestion of the tubulin alpha, beta heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin-cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C-terminus of the molecule. The C-terminal peptides of both alpha and beta subunits of tubulin were purified by anion-exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp-438 and His-406 of alpha and Gln-433 and His-396 of beta tubulin chains. Quantitative analysis of our data show that cleavage at positions His-406 (alpha) and His-396 (beta) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu-445 or -435 of alpha and beta tubulin, respectively.

The X-ray repair cross-complementing group 1 (XRCC1) and xeroderma pigmentosum group D (XPD) genes are involved in base excision repair and nucleotide excision repair of DNA repair pathways, respectively. A growing body of evidence suggests that XRCC1 and XPD are important in environmentally induced cancers, and polymorphisms in both genes have been identified. To determine whether the XRCC1 (codon Arg399Gln) and XPD (codon Asp312Asn and codon Lys751Gln) polymorphisms are associated with prostate cancer susceptibility, we genotyped these polymorphisms in a primarily Caucasian sample of 506 sibships (n = 1,117) ascertained through a brother with prostate cancer. Sibships were analyzed with a Cox proportional hazards model with age at prostate cancer diagnosis as the outcome. Of the three polymorphisms investigated, only the XPD codon 312 Asn/Asn genotype had an odds ratio (OR) significantly different from one (OR, 1.61; 95% CI, 1.03-2.53). Analyses stratified by the clinical characteristics of affected brothers in the sibship did not reveal any significant heterogeneity in risk. In exploring two-way gene interactions, we found a markedly elevated risk for the combination of the XPD codon 312 Asn/Asn and XRCC1 codon 399 Gln/Gln genotypes (OR, 4.81; 95% CI, 1.66-13.97). In summary, our results suggest that the XPD codon 312 Asn allele may exert a modest positive effect on prostate cancer risk when two copies of the allele are present, and this effect is enhanced by the XRCC codon 399 Gln allele in its recessive state.

Mechanisms behind the strong associations of esophageal adenocarcinoma risk with gastroesophageal reflux (GOR) and body mass remain to be defined. In a nationwide population-based case-control study, we examined associations of polymorphisms in the DNA repair genes XPD, XPC, XRCC1 and XRCC3 with risk of esophageal adenocarcinoma, squamous-cell carcinoma (SCC) and gastric cardia adenocarcinoma, and paid special attention to possible interactions with symptomatic reflux or body mass. We collected blood samples from 96, 81 and 126 interviewed incident cases of esophageal adenocarcinoma, esophageal SCC and gastric cardia adenocarcinoma, respectively, and 472 randomly selected controls, frequency-matched with regard to age and sex. DNA was extracted and polymorphisms in XPD codon 751 (Lys->>Gln), codon 312 (Asp->>Asn), C insertion in intron 10 of XPD, XPC codon 939 (Lys->>Gln), XRCC1 codon 399 (Arg->>Gln) and XRCC3 codon 241 (Thr->>Met) were examined using PCR-RFLP. Odds ratios (ORs) derived from multivariate logistic regression with adjustments for potential confounding factors estimated relative risks. XPD codon 751 Lys/Gln and Gln/Gln genotypes, compared with Lys/Lys genotype, were both associated with a more than doubled risk for esophageal adenocarcinoma (OR=2.4; 95% CI=1.4-4.4; OR=2.7, 95% CI=1.3-5.9). The combined effects of these genotypes and symptomatic GOR or body mass showed borderline significant deviation from additivity. Excess risks for esophageal SCC were also noted for XPD 751Gln variant genotypes. Other studied variants were not found to be related to the three tumors. Our study suggests that XPD 751Gln allele is a potential genetic marker for susceptibility to esophageal adenocarcinoma.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.

TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine (m(5)U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-A resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA-RNA complex for crystallization. The protein-RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58-G57-C56-U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the m(5)U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates.

Enantiopure heterocyclic Boc-protected Phe-Gly dipeptidomimetics containing 1,3,4-oxadiazole, 1,2,4-oxadiazole, and 1,2,4-triazole ring systems have been synthesized as building blocks in the synthesis of pseudopeptides. Three derivatives (1-3) have the carboxylic acid function directly bound to the heterocyclic ring, and three derivatives (4-6) have an extra methylene group between the heterocyclic ring and the acid function to allow for an increased conformational flexibility. The mimetics were used as Phe-Gly replacements in the biologically active peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) and substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH(2), SP). The pseudopeptide synthesis was performed using solid-phase methodology on a MBHA-resin using Boc-chemistry. The biological evaluation was performed by testing the micro- and delta-opioid receptor affinities of the dermorphin pseudopeptides and the NK(1) receptor affinities of the SP pseudopeptides. The results showed that all mimetics except 3 were excellent replacements of Phe-Gly in dermorphin since they displayed affinities for the micro-receptor (IC(50) = 12-31 nM) in the same range as dermorphin itself (IC(50) = 6.2 nM). The agonist activity of three pseudopeptides at human micro-receptors was also evaluated. It was shown that the tested compounds retained their agonist activity. The SP pseudopeptides showed considerably lower affinities (IC(50)>> 1 microM) for the NK(1) receptor than SP itself (IC(50) = 1.5 nM) indicating that the Phe-Gly replacements prevent the pseudopeptides from adopting bioactive conformations.

We previously reported a new brain-specific protein with a molecular mass of 14 kDa, specifically present in synapses around neurons but not in glial cells [Nakajo, S., Omata, K., Aiuchi, T., Shibayama, T., Okahashi, I., Ochiai, H., Nakai, Y., Nakaya, K. & Nakamura, Y. (1990) J. Neurochem. 55, 2031-2038]. In the present study, we determined the primary structure of this protein, found that it is phosphorylated in vitro and in vivo, and designated it phosphoneuroprotein 14 (PNP 14). The protein is a single polypeptide with 134 amino acid residues (molecular mass = 14122 Da), and it contains a hydrophobic region at the center of the molecule. The carboxy-terminal region has all seven proline residues, and is rich in glutamic acid, which contribute to the acidic property of the protein. The amino-terminal region possesses four unique repetitive motifs, Glu(Ser)-Lys-Thr-Lys-Glu(Gln)-Gly(Gln)-Val(Ala). When a cytosolic fraction prepared from rat cerebral cortex was incubated with [gamma-32P]ATP, 32P was incorporated into PNP 14. Phosphorylated PNP 14 was immunoprecipitated from rat brain synaptosomes labeled metabolically with [32P]orthophosphate. Injection of [32P]orthophosphate into the third ventricle of rat brain resulted in incorporation of radioactive phosphate into PNP 14. We have also found that Ca2+, calmodulin-dependent protein kinase II phosphorylates serine residue(s) of PNP 14 in vitro. The results suggest that PNP 14 may be important to neuronal cells, but not to glial cells, and that its physiological functions may be controlled by the phosphorylation reaction.

This review focuses on the role of acute pH changes in the regulation of Gln/Glu metabolism in the kidney, liver, and brain. Alterations of proton concentration ([H(+)]) profoundly affect flux through phosphate-dependent glutaminase (PDG) or glutamate dehydrogenase (GDH), the primary enzymes responsible for mitochondrial metabolism of glutamine and glutamate, respectively. In the kidney, acute acidosis stimulates Gln uptake and its metabolism via the PDG pathway. The Glu formed from Gln can be removed via 1) oxidative deamination through the GDH reaction, 2) transamination reactions, and 3) transport of Glu from intracellular to extracellular compartment, thereby diminishing the intramitochondrial pool of glutamate sufficiently to stimulate flux through the PDG pathway. Converse changes may occur with increased pH. In the liver, acidosis diminishes the rate of Gln and Glu metabolism via the PDG and GDH pathways, but stimulates glutamine synthesis (i.e., glutamine recycling). Alkalosis has little effect. Hepatic Gln metabolism via the PDG pathway has a central role in ureagenesis via 1) supplementation of nitrogen for the synthesis of carbamyl phosphate, and 2) providing glutamate for N-acetylglutamate synthesis. In the brain, Gln/Glu metabolism links ammonia detoxification and energy metabolism via 1) detoxification of ammonia and excess glutamate by glutamine synthesis in astrocytes, 2) formation and export of glutamine to neurons where it is metabolized to glutamate and GABA, and 3) production of alpha-ketoglutarate and lactate from Glu and their transport to neurons. Changes in intracellular pH associated with changes in cellular [K(+)] may have a key role in the regulation of these processes of glial-neuronal metabolism of Gln/Glu metabolism.

A novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. Activity is then partially restored by substrates containing the missing catalytic functional group. Replacement of the catalytic His with Ala in the Bacillus amyloliquefaciens subtilisin gene (the mutant is designated His64Ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/Km) by a factor of a million when assayed with N-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA). Model building studies showed that a His side chain at the P2 position of a substrate bound at the active site of subtilisin could be virtually superimposed on the catalytic His side chain of this serine protease. Accordingly, the His64Ala mutant hydrolyzes a His P2 substrate (sFAHF-pNA) up to 400 times faster than a homologous Ala P2 or Gln P2 substrate (sFAAF-pNA or sFAQF-pNA) at pH 8.0. In contrast, the wild-type enzyme hydrolyzes these three substrates with similar catalytic efficiencies. Additional data from substrate-dependent pH profiles and hydrolysis of large polypeptides indicate that the His64Ala mutant enzyme can recover partially the function of the lost catalytic histidine from a His P2 side chain on the substrate. Such "substrate-assisted catalysis" provides a new basis for engineering enzymes with very narrow and potentially useful substrate specificities. These studies also suggest a possible functional intermediate in the evolution of the catalytic triad of serine proteases.

A peptide corresponding to the three zinc finger domains of the human transcription factor Sp1 has been expressed and found to bind a consensus Sp1 binding site with the sequence 5'-GGGGCGGGG-3'. Examination of the amino acid distributions within a large zinc finger sequence data base and chemical arguments suggested that a particular Arg to Gln sequence change might convert binding specificity to 5'-GGGGCAGGG-3'. Experimental tests of this hypothesis revealed that such a change could be induced only when two other sequence changes, deduced from examination of sequence correlations, were made as well. These results provide the most direct information to date about how zinc finger proteins might recognize adenine-containing binding sites and bear on the existence and nature of any code between zinc finger protein and binding site sequences.

Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-(13)C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [(13)C, (1)H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 (13)C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 micromol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 micromol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variations. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants. One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (codon 94) and a separate G----A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions: Thr for Ile (codon 114) and Arg for Lys (codon 268). Heterozygosity was detected in three NAT2 samples: two were heterozygous for the rapid and one of the allelic variants, and the third was a compound heterozygote of both mutant alleles. The results show conclusively that the genetically variant NAT is encoded by NAT2.

Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. The mutation Ala----Thr in this triplet drastically reduced transmission, whereas the mutation Asp----Asn had no effect, and the mutation Asp----Lys consistently reverted to the wild-type residue. The mutation Lys----Glu, in the residue adjacent to the glycine of the triplet, drastically reduced transmission, whereas the mutation Gln----Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence of a glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility.

Acetyl-L-carnitine (ALCAR) is an endogenous metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane. Exogenously administered ALCAR has been used as a nutritional supplement and also as an experimental drug with reported neuroprotective properties and effects on brain metabolism. The aim of this study was to determine oxidative metabolism of ALCAR in the immature rat forebrain. Metabolism was studied in 21-22 day-old rat brain at 15, 60 and 120 min after an intraperitoneal injection of [2-(13)C]acetyl-L-carnitine. The amount, pattern, and fractional enrichment of (13)C-labeled metabolites were determined by ex vivo(13)C-NMR spectroscopy. Metabolism of the acetyl moiety from [2-(13)C]ALCAR via the tricarboxylic acid cycle led to incorporation of label into the C4, C3 and C2 positions of glutamate (GLU), glutamine (GLN) and GABA. Labeling patterns indicated that [2-(13)C]ALCAR was metabolized by both neurons and glia; however, the percent enrichment was higher in GLN and GABA than in GLU, demonstrating high metabolism in astrocytes and GABAergic neurons. Incorporation of label into the C3 position of alanine, both C3 and C2 positions of lactate, and the C1 and C5 positions of glutamate and glutamine demonstrated that [2-(13)C]ALCAR was actively metabolized via the pyruvate recycling pathway. The enrichment of metabolites with (13)C from metabolism of ALCAR was highest in alanine C3 (11%) and lactate C3 (10%), with considerable enrichment in GABA C4 (8%), GLN C3 (approximately 4%) and GLN C5 (5%). Overall, our (13)C-NMR studies reveal that the acetyl moiety of ALCAR is metabolized for energy in both astrocytes and neurons and the label incorporated into the neurotransmitters glutamate and GABA. Cycling ratios showed prolonged cycling of carbon from the acetyl moiety of ALCAR in the tricarboxylic acid cycle. Labeling of compounds formed from metabolism of [2-(13)C]ALCAR via the pyruvate recycling pathway was higher than values reported for other precursors and may reflect high activity of this pathway in the developing brain. This is, to our knowledge, the first study to determine the extent and pathways of ALCAR metabolism for energy and neurotransmitter biosynthesis in the brain.

OBJECTIVE

Interindividual differences in DNA repair capacity not only modify individual susceptibility to carcinogenesis, but also affect individual response to cancer treatment. Nucleotide excision repair (NER) is one of the major DNA repair pathways in mammalian cells involved in the removal of a wide variety of DNA lesions. Polymorphisms in NER genes may influence DNA repair capacity and affect clinical outcome of bladder cancer treatment.

METHODS

To test the influence of NER gene polymorphisms on superficial bladder cancer outcome (recurrence and progression), we conducted a follow-up study of 288 patients with superficial bladder cancer. Median follow-up among patients who were recurrence-free at the end of observation was 21.7 months from diagnosis. The specific polymorphic loci examined include XPA [A/G at 5' untranslated region (UTR)], XPC (poly AT, Ala(499)Val, Lys(939)Gln), XPD (Asp(312)Asn, Lys(751)Gln), XPG (His(1104)Asp), ERCC 1 (G/T at 3' UTR), and ERCC6 (Met(1097)Val, Arg(1230)Pro).

RESULTS

The ERCC6 (Met(1097)Val) polymorphism had a significant impact on recurrence: carriers of at least one variant allele (Val) had a significantly higher recurrence risk than carriers of the wild-type allele (Met/Met; hazard ratio, 1.54; 95% confidence interval, 1.02-2.33). There were no overall statistically significant differences in the distributions of the other polymorphisms between patients with and without recurrence. However, when we combined these variant genotypes, there was a significant trend for an increased recurrence risk with an increasing number of putative high-risk alleles. Using individuals with five or fewer putative high-risk alleles as the reference group, individuals with six to seven risk alleles and individuals with eight or more risk alleles had higher recurrence risks, with hazard ratios of 0.92 (0.54-1.57) and 2.53 (1.48-4.30), respectively (P for trend < 0.001). There was also a significant trend for shorter recurrence-free survival time with increasing number of variant alleles (log rank test, P = 0.0007). When we stratified the patients according to intravesical Bacillus Calmette-Guerin treatment, we found a significant trend for shorter recurrence-free survival time in patients with variant alleles of XPA or ERCC6 polymorphisms who received Bacillus Calmette-Guerin treatment (log rank test, P = 0.078 and 0.022, respectively). There were no significant individual or joint associations between these polymorphisms and progression.

CONCLUSIONS

These data suggest that interindividual differences in DNA repair capacity may have an important impact on superficial bladder cancer recurrence. A pathway-based approach is preferred to study the effects of individual polymorphism on clinical outcomes.

Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, light-oxygen-voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jalpha helix and, ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue [Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain] switches its hydrogen bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Ibeta strand that interacts with the Jalpha helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jalpha helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudodark state (Q513L) or a pseudolit state (Q513N). Further, both mutations changed the photochemical properties of this receptor, in particular the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the Ibeta strand to the peripheral Jalpha helix.