Search for the rare decays K(L)→π0π0µ+µ- and K(L)→π0π0X0→π0π0µ+µ-.

The KTeV E799 experiment has conducted a search for the rare decays, K(L)→π(0)π(0)µ(+)µ(-) and K(L)→π(0)π(0)X(0)→π(0)π(0)µ(+)µ(-), where the X(0) is a possible new neutral boson that was reported by the HyperCP experiment with a mass of (214.3 ± 0.5) MeV/c(2). We find no evidence for either decay. We obtain upper limits of Br(K(L)→π(0)π(0)X(0)→π(0)π(0)µ(+)µ(-)) < 1.0 × 10(-10) and Br(K(L)→π(0)π(0)µ(+)µ(-)) < 9.2 × 10(-11) at the 90% confidence level. This result rules out the pseudoscalar X(0) as an explanation of the HyperCP result under the scenario that the dsX(0) coupling is completely real.

Search for lepton-flavor-violating decays of the neutral kaon.

The Fermilab KTeV experiment has searched for lepton-flavor-violating decays of the K(L) meson in three decay modes. We observe no events in the signal region for any of the modes studied, and we set the following upper limits for their branching ratios at the 90% C.L.: BR(K(L) --> pi(0) micro(+/-) e(-/+)) <7.6 x 10(-11); BR(K(L) --> pi(0)pi(0) micro(+/-) e(-/+)) <1.7 x 10(-10); BR(pi(0) --> micro(+/-) e(-/+)) <3.6 x 10(-10). This result represents a factor of 82 improvement in the branching ratio limit for K(L) --> pi(0) micro(+/-) e(-/+) and is the first reported limit for K(L) --> pi(0)pi(0) micro(+/-) e(-/+).

Determination of the parity of the neutral pion via its four-electron decay.

We present a new determination of the parity of the neutral pion via the double Dalitz decay pi0-->e+e-e+e-. Our sample, which consists of 30,511 candidate decays, was collected from KL-->pi0pi0pi0 decays in flight at the KTeV-E799 experiment at Fermi National Accelerator Laboratory. We confirm the negative pi0 parity and place a limit on scalar contributions to the pi0-->e+e-e+e- decay amplitude of less than 3.3% assuming CPT conservation. The pi0gamma*gamma* form factor is well described by a momentum-dependent model with a slope parameter fit to the final state phase-space distribution. Additionally, we have measured the branching ratio of this mode to be B(pi0-->e+e-e+e-)=(3.26+/-0.18)x10(-5).

Measurements of the decay KL-->e+ e- gamma.

The E799-II (KTeV) experiment at Fermilab has collected 83 262 K(L)-->e+ e- gamma(gamma) events above a background of 79 events. We measure a decay width, normalized to the K(L)-->pi0pi0pi(D)0 (pi0-->gammagamma, pi0-->gammagamma, pi(D0-->e+ e- gamma(gamma)) decay width, of Gamma(K(L)-->e+e-gamma(gamma))/Gamma(K(L)-->pi0pi0pi(D)0)=(1.3302+/-0.0046(stat)+/-0.0102(syst)) x 10(-3). We also measure parameters of two K(L)gamma*gamma form factor models. In the Bergström-Massó-Singer parametrization, we find Calpha(K*)= -0.517 +/- 0.030(stat) +/- 0.022(syst). We separately fit for the first parameter of the D'Ambrosio-Isidori-Portolés model and find alpha(DIP)= -1.729 +/- 0.043(stat) +/- 0.028(syst).

First observation of K{L}-->pi{+/-}e{-/+}nue{+}e{-}.

This Letter is the first report of the K{L}-->pi{+/-}e{-/+}nue{+}e{-} decay. Based on 19 208+/-144 events, we determine the branching fraction, B(K{L}-->pi{+/-}e{-/+}nue{+}e{-}M_{e{+}e{-}}>5 MeV/c{2},E{e{+}e{-}}{*}>30 MeV)=(1.285+/-0.041)x10{-5}, and Gamma(K{e3ee}M{e{+}e{-}}>5 MeV/c{2})/Gamma(K{e3})=[4.57+/-0.04(stat)+/-0.14(syst)]x10{-5}. This ratio agrees with a theoretical prediction based on chiral perturbation theory (ChPT) calculated to O(p{4}). The measured kinematical distributions agree with those predicted by just ChPT O(p{4}), but show significant disagreement with ones predicted by leading-order ChPT.

Measurement of the K0 charge radius and a CP-violating asymmetry and a search for CP-violating E1 direct photon emission in the rare decay KL--> pi+ pi- e+ e-.

Using the complete KTeV data set of 5,241 candidate K(L)--> pi(+) pi(-) e(+) e(-) decays (including an estimated background of 204 +/- 14 events), we have measured the coupling g(CR)= 0.163 +/- 0.0149(stat) +/- 0.023(syst) of the CP conserving charge radius process and from it determined a K(0) charge radius of = [-0.077 +/- 0.007(stat) +/- 0.011(syst)]fm(2). We have determined a first experimental upper limit of 0.04 (90% C.L.) /g(e1)/ / /g(M1)/ of the couplings for the E1 and M1 direct photon emission processes. We also report the measurement of /g(M1)/ including a vector form factor /g(M1)/(1 + (a(1)/a(2))/((M(2)(p)-(M(2)(k))= 2M(K)E(gamma*)), where vector /g(M1)/= 1.11+/- 0.12(stat) +/- 0.08(syst) and a(1)/a(2) = [-0.744 +/- 0.027(stat) +/- 0.032(syst)] GeV(2)/c(2). Finally, a CP-violating asymmetry of [13.6 +/- 1.4(stat) +/- 1.5(syst)]% in the CP and T odd angle phi between the decay planes of the e(+) e(-) and pi(+) pi(-) pairs in the K(L) center of mass is reported.

Observation of the decay xi0 --> sigma+ mu- nu(mu).

The xi0 muon semileptonic decay has been observed for the first time with nine identified events using the KTeV beam line and detector at Fermilab. The decay is normalized to the xi0 beta decay mode and yields a value for the ratio of decay rates gamma(xi0 --> sigma+ mu- nu(mu))/gamma(xi0 --> sigma+ e- nu(e)) of [1.8(-0.5)(+0.7)(stat) +/- 0.2(syst)] x 10(-2). This is in agreement with the SU(3) flavor symmetric quark model.

A sulfhydryl oxidase from chicken egg white.

A dimeric glycoprotein containing one FAD per approximately 80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a Km of 150 microM, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH2) showing a charge transfer band (lambdamax 560 nm; epsilonobs 2.5 mM-1 cm-1). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH2, but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH2-like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.

Sudden death of chicken embryos with hereditary riboflavin deficiency.

Riboflavin-binding protein (RfBP) mediates the deposition of riboflavin during the formation of eggs in birds. Hens of a strain of Single-Comb White Leghorn chickens, which are genetically unable to produce RfBP, lay eggs containing insufficient riboflavin to sustain embryogenesis beyond 13 or 14 d of incubation. Embryos in these eggs grow normally until the day of death, and their heart rate is normal to within an hour of death. The effects of riboflavin-deficiency first appear after d 10 of incubation when embryos become severely hypoglycemic and begin to accumulate intermediates of fatty acid oxidation. Although the activities of flavin-dependent enzymes are reduced generally, the 80% reduction in the activity of medium-chain acyl-CoA dehydrogenase further suggests that the major metabolic consequence of riboflavin deficiency is a severe impairment of fatty acid oxidation. The riboflavin-deficient strain provides numerous insights into the metabolism of normal hens and chicken embryos and may be a useful model for sudden death syndromes in humans.

An oocytic membrane receptor for biotin-binding protein.

The chicken oocyte accumulates a biotin-binding protein (BBP) in the yolk that is distinct from the avidin in the 'egg white'. An identical BBP to that of the yolk is also present in the circulation of the laying hen. We report the first evidence for the existence of a BBP receptor in the oocyte vitelline membrane. Reduction of the 100 kDa receptor results in loss of BBP-binding activity; this suggests that a disulfide bonded region of the receptor is necessary for maintaining BBP-binding activity. We show further that the levels of serum BBP are strictly dependent on the presence of estrogen. As expected, BBP is not detected in significant quantities in rooster serum. Thus, these results suggest that circulatory BBP, like other estrogen-dependent components of serum, has a cognate binding activity on the oocyte membrane that may mediate its endocytosis.

Riboflavin-binding protein induces early death of chicken embryos.

Maternal riboflavin-binding protein (RfBP) mediates the deposition of riboflavin in avian eggs. A strain of Single-Comb White Leghorn chicken, genetically unable to synthesize RfBP, produces eggs deficient in riboflavin. Embryos in these eggs die on or about 13 of incubation, with severe hypoglycemia and impaired fatty acid oxidation. Injection of free riboflavin prior to incubation allows these embryos to survive and hatch normally. The egg whites of eggs from the RfBP-deficient strain were injected with different relative amounts of riboflavin and RfBP to determine their effects on the developing embryo. Injection of protein-bound riboflavin resulted in a substantial number of the embryos (27%) surviving until the end of the experiment (d 19) with an average weight of 13.7 g, demonstrating that some embryos were able to use the bound riboflavin. In control groups injected with riboflavin alone, 10 of 17 embryos survived until d 19 with an average weight 18.5 g, whereas those injected with saline died between d 10 and 13 with an average weight of 3.8 g. A fourth group, injected with apo-RfBP, produced embryos with an average weight of 0.24 g. This early death suggests that the protein scavenges the small amount of riboflavin normally available to the early embryo. Further studies showed that titration of RfBP-binding sites with exogenous riboflavin lessens this effect. These studies show that although riboflavin-binding protein is essential for the deposition of riboflavin in eggs, its presence in excess can diminish the availability of riboflavin to the chicken embryo.

Riboflavin-deficient chicken embryos: hypoglycemia without dicarboxylic aciduria.

Chicken embryos in eggs laid by hens that are genetically unable to deposit riboflavin into their eggs die on or about the 13th day of incubation. We show that these riboflavin-deficient embryos grow normally until the day of death and that their heart rate is normal to within an hour of death. The embryos have symptoms of impaired fatty acid oxidation, including decreased activity of FAD-dependent medium-chain acyl CoA dehydrogenase in liver and heart along with a significant accumulation of intermediates of fatty acid oxidation (C10, C12, and C14 acids). Unlike riboflavin-deficient mammals, the embryos do not accumulate dicarboxylic acids derived from omega-oxidation of fatty acids. Blood glucose is near normal on day 10 but declines to undetectable levels by the time of death. Allantoic fluid from the riboflavin-deficient embryos of 11 days or older contains more lactate than 3-hydroxybutyrate, while in normal embryos the reverse is true. No appreciable amounts of glycine-conjugated acids were found. We conclude that the major and perhaps primary pathological effect of riboflavin deficiency in chicken embryos is the impairment of fatty acid beta-oxidation, and that the subsequent depletion of limited carbohydrate reserves leads to sudden death.

Folate metabolism and deposition in eggs by laying hens.

White Leghorn hens were fed purified folate-deficient diets or commercial corn- and soybean meal-based diets supplemented with different amounts of folic acid. The folate contents of egg yolk and blood plasma from these hens were estimated with an isotope-dilution, radioligand-binding assay. Folates in egg yolk were concentrated approximately 43-fold relative to the blood plasma from which they were derived. Yolk and plasma folate concentrations became saturated with increasing dietary folate. Hens fed a commercial, folate-sufficient diet (0.72 mg folate/kg) produced eggs with slightly less than half of the maximal folate content. Based on tritium deposition in egg yolk and egg white, the biological half-life of [3H]folic acid injected intraperitoneally into two folate-sufficient hens was approximately 15 days, while it was > or = 40 days in two hens fed a purified folate-deficient diet (0.07 mg folate/kg) that also reduced egg production. Radioactivity in egg yolk was concentrated more than 100-fold relative to egg white in both cases. The [3H]folates remaining in the hens at the end of the experiment were substantially more concentrated in liver than in kidney, heart, or skeletal muscle. The specific radioactivity of folates in the liver of folate-deficient hens after 78 days was almost 10 times greater than in folate-sufficient hens after 39 days. Laying hens have highly efficient conservation and delivery systems for folates.

Riboflavinuria in the rd chicken. 5'-splice site mutation in the gene for riboflavin-binding protein.

Eggs of oviparous species must contain all components required for normal embryonic development. Among these, the vitamin riboflavin is deposited into egg white and yolk bound to the carrier protein, riboflavin-binding protein (ribBP). We have begun to investigate the mechanisms underlying ribBP transport pathways by molecular characterization of a relevant mutation in chicken. The autosomal recessive rd allele in Gallus gallus domesticus prevents the synthesis of functional ribBP and induces embryonic death on day 13. Polymerase chain reaction primers derived from the previously determined wild type cDNA sequence were used to amplify and clone cDNAs for ribBP from normal (Rd) and deficient (rd) animals. The rd allele was associated with a 100-nucleotide deletion in the messenger RNA for ribBP. Genomic clones were generated via polymerase chain reaction using primers flanking this 100-base pair deletion, and the resulting 2.1-kilobase pair clones were sequenced. The deletion in the rd ribBP cDNA corresponds precisely to an exon. The splice site following this exon contains a G-->A mutation at position 1 of the downstream 5'-splice donor sequence. The effect of this anomaly and the cause of the rd phenotype is the loss of the 100-base pair exon during the splicing process.

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.

Separation and characterization of the two Asn-linked glycosylation sites of chicken serum riboflavin-binding protein. Glycosylation differences despite similarity of primary structure.

Serum riboflavin-binding protein, a phosphoglycoprotein from the blood of laying hens, contains two Asn-Xaa-(Thr)Ser sequons in very similar but well-separated regions of amino acid sequence. In order to evaluate the effect of local amino acid sequence on the structure of the attached oligosaccharides, serum riboflavin-binding protein was purified to homogeneity, reduced and alkylated, digested with trypsin, and the two glycopeptides were separated by reversed-phase chromatography. After digestion with peptide-N-glycosidase F the released oligosaccharides were separated by high-pH anion-exchange chromatography and the oligosaccharide profiles of the two glycopeptides were compared. Although the two asparagine residues that are glycosylated are contained in pentapeptide segments in which four out of five amino acids are identical, the array of oligosaccharides present at each site show differences in both type and distribution. This suggests that local secondary or tertiary structure, or the order of glycosylation, influences the oligosaccharide structure more than does the primary structure flanking the attachment site.

Effect of riboflavin-binding protein deficiency on riboflavin metabolism in the laying hen.

Normal chicken eggs contain substantial amounts of riboflavin, all of which is bound to a specific, high-affinity, riboflavin-binding protein (RfBP). Two hens, genetically unable to produce RfBP and thus unable to deposit sufficient riboflavin in their eggs, were compared to two normal hens with respect to the biological half-life of [14C]riboflavin, the tissue distribution of 14C-labeled flavins, and the relative contributions of tissue and dietary riboflavin to flavins deposited in the egg. The biological half-life of [14C]riboflavin was slightly but insignificantly less in the RfBP-deficient hens (11.5 +/- 1.7 days vs 15.1 +/- 3.3 days). The 14C-labeled flavin content of a variety of tissues 3 weeks after the intraperitoneal injection of 5 microCi of riboflavin was also very similar among the four hens. In contrast, the 14C-labeled flavin content of egg yolk, egg albumen, and blood plasma from RfBP-deficient birds was less than 10% of normal. For all hens, the specific radioactivity of flavins in yolk and albumen was similar to that in liver but less than that in heart. We conclude that riboflavin deposited in egg had equilibrated with the large hepatic flavin pool and was not derived preferentially from unlabeled dietary riboflavin. Other than the inability to deposit riboflavin in their eggs, hens of the mutant strain have normal riboflavin metabolism.

Competitive binding assays for high-affinity binders in the presence of endogenous ligands: application to biotin-binding proteins.

Endogenous ligands complicate radioligand-binding assays of high-affinity binding proteins by obscuring binding sites or by diluting the labeled ligand. We have developed a mathematical model for such systems where radioligand and endogenous ligand are structurally identical. Data which relate radioligand binding at equilibrium as a function of sample volume can be plotted such that the concentrations of endogenous ligand and binder are graphically determined; however, a more precise determination may be done by nonlinear regression with the aid of a microcomputer. The method is demonstrated for the assay of biotin-binding proteins in the presence of a range of endogenous biotin concentrations below and above that required to saturate the binding sites.

Biotin diffusion in stored chicken eggs: a re-assessment.

1. The possibility that biotin in stored eggs migrates from the yolk into the albumen was re-investigated. 2. Sets of eggs collected from 12 Single Comb White Leghorn hens were stored at 5, 8, 12, 15, or 18 degrees C for 5, 10, 15, 20, or 25 days and then frozen. 3. For each set, yolk and albumen samples adjacent to the yolk membrane were each pooled and assayed for protein-bound biotin by radioligand exchange with non-linear regression analysis of the data. 4. In contrast to previous work reported by Bush and White (Comp. Biochem. Physiol. 93B, 543-547, 1989), no evidence of biotin diffusion was found. 5. Non-linear regression was used to re-evaluate the data from the earlier study, confirming the results, although less strikingly. 6. The discrepancy is partly due to the method of data analysis, although differences in breed or number of hens may also be contributing factors.