Spaced training improves learning in Ts65Dn and Ube3a mouse models of intellectual disabilities.

Benefits of distributed learning strategies have been extensively described in the human literature, but minimally investigated in intellectual disability syndromes. We tested the hypothesis that training trials spaced apart in time could improve learning in two distinct genetic mouse models of neurodevelopmental disorders characterized by intellectual impairments. As compared to training with massed trials, spaced training significantly improved learning in both the Ts65Dn trisomy mouse model of Down syndrome and the maternally inherited Ube3a mutant mouse model of Angelman syndrome. Spacing the training trials at 1 h intervals accelerated acquisition of three cognitive tasks by Ts65Dn mice: (1) object location memory, (2) novel object recognition, (3) water maze spatial learning. Further, (4) spaced training improved water maze spatial learning by Ube3a mice. In contrast, (5) cerebellar-mediated rotarod motor learning was not improved by spaced training. Corroborations in three assays, conducted in two model systems, replicated within and across two laboratories, confirm the strength of the findings. Our results indicate strong translational relevance of a behavioral intervention strategy for improving the standard of care in treating the learning difficulties that are characteristic and clinically intractable features of many neurodevelopmental disorders.

Origins of high sequence selectivity: a stopped-flow kinetics study of DNA/RNA hybridization by duplex- and triplex-forming oligonucleotides.

Stopped-flow UV kinetics and thermal denaturation experiments are used to examine the origins of high sequence selectivity and binding affinity of circular triplex-forming oligonucleotides with single-stranded DNA/RNA targets. These 34-nt probes are hybridized to a series of 12-nt target sequences which are fully complementary or which contain a single mismatch. Also studied for comparison are standard 12-nt Watson-Crick DNA or RNA complements. Several novel findings are described: (1) Circular triplex-forming oligomers bind targets with very high thermodynamic selectivity (up to 8-10 kcal/mol against a single-nucleotide mismatch), while linear strands show only 2-3 kcal/mol selectivity. (2) Rates for triplex formation by circular ligands are much greater than other reported triplex formation modes and are nearly the same as for Watson-Crick duplex formation. (3) DNA-DNA and RNA-RNA hybridization rates are similar for both duplex and triplex formation. (4) For both modes of binding, hybridization rates do not vary when a mismatch is introduced into the target, and, therefore, binding selectivity is reflected in large variations in dissociation, rather than association rates. Finally, (5) binding selectivity of circular ligands becomes significantly greater as pH is lowered; results indicate that the high sequence selectivity of the circular DNA ligand is due in large part to the special stability of the protonated C+G-C triad relative to unprotonated mismatched triads. The results are useful in the understanding of properties of nucleic acid complexes in general and give insight into optimum design for synthetic DNA-binding ligands.

Horseradish peroxidase Phe172-->Tyr mutant. Sequential formation of compound I with a porphyrin radical cation and a protein radical.

A gene coding for the F172Y mutant of horseradish peroxidase isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperda (SF-9) and Trichoplusia ni egg cell homogenate (HighFive) cells. Homology modeling with respect to three peroxidases for which crystal structures are available places Phe172 on the proximal side of the heme in the vicinity of porphyrin pyrrole ring C. The pH optimum and spectroscopic properties of the F172Y mutant are essentially identical to those of wild type HRP. Vmax values show that the mutant protein retains most of the guaiacol oxidizing activity. Stopped flow studies indicate that Compound I is formed with H2O2 at the same rate (kappa 1 = 1.6 x 10(7) M-1 s-1) at both pH 6.0 and 8.0 as it is with the wild type enzyme. This Compound I species decays rapidly at a rate kappa 2 = 1.01 s-1, pH 7.0, to a second two-electron oxidized species that retains the ferryl (FeIV = O) absorption. EPR studies establish that a ferryl porphyrin radical cation is present in the initial Compound I, but electron transfer from the protein results in formation of a second Compound I species with an unpaired electron on the protein (presumably on Tyr172). The presence or absence of oxidizable amino acids adjacent to the heme is thus a key determinant of whether the second oxidation equivalent in Compound I is found as a porphyrin or protein radical cation.

Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation.

Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:a-2',3':c]-phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.

Luminescence of ruthenium(II) polypyridyls: evidence for intercalative binding to Z-DNA.

Photophysical studies have been undertaken to characterize the binding interactions of enantiomers of Ru(phen)3(2+), Ru(DIP)3(2+), and racemic Ru(bpy)2dppz2+ (where phen = 1,10-phenanthroline, DIP = 4,7-diphenylphenanthroline, and dppz = dipyridophenazine) with Z-form poly d(GC). Parallel enhancements in steady state luminescent intensity and a lengthening of luminescent lifetimes are seen for ruthenium enantiomers with Z-DNA as for B-DNA but with enantioselectivities reversed. Greater enhancements are seen for delta-isomers with the right-handed helix but for lambda-isomers with the left-handed helix. Ru(bpy)2dppz2+, an avid intercalator in B-DNA, displays no luminescence free in aqueous solution, but luminesces brightly bound to either B- or Z-poly d(GC). Stern-Volmer quenching studies also support the enantioselective preference in binding to B-DNA by delta-isomers and a reversal with binding to Z-DNA preferentially by the lambda-isomers. Steady state polarization studies indicate a rigid association of the complexes with both B- and Z-DNA on the time-scale of their emission and again with symmetrical enantioselectivities for the left and right-handed helices. Given the well characterized intercalative association of the complexes with B-DNA, the parallel results seen here with Z-DNA point strongly to a comparable intercalative association with the Z-form helix. That molecules may interact with Z-DNA through intercalation has not been demonstrated previously and now requires consideration in describing the range of interactions of small molecules and proteins with Z-DNA.

Evaluation of human viral disease transmission through plasma products.

Alpha's Wet Heat-Treatment process is being applied to both Factor VIII (AHF) and Factor IX Complex (PTC). Twelve hemophilia A, five hemophilia B, and one von Willebrands patient have been followed for at least 6 months for evidence of non-A, non-B hepatitis. No ALT elevations were seen in the hemophilia B patients. There have been four cases of ALT elevation, three in hemophilia A patients and one in the von Willebrand's patient. A subset of these patients have been followed for over one year for anti-HTLV-III status. No patient, either hemophilia A, hemophilia B, or von Willebrand's seroconverted to anti-HTLV-III positive status. Intravenous gamma globulin was studied in 11 normal patients given a single infusion and in 23 immune deficient patients with multiple infusions and evaluations of liver enzymes over a two year period. No elevated ALT or AST values were seen in either group.

Evaluation of two viral inactivation methods for the preparation of safer factor VIII and factor IX concentrates.

We report here the results of our evaluation of two procedures to eliminate viruses in factor VIII and factor IX coagulation factor concentrates. Both procedures were equally effective in the in vitro destruction of marker viruses. However, in a controlled infectivity test in chimpanzees, treatment at 60 degrees C for 20 hours inactivated greater than 500 and less than 10,000 chimpanzee infectious doses (CID) of hepatitis B virus, while treatment at 98 degrees C for 30 minutes inactivated less than 500 CID. Both methods were successful in preventing infection with an undetermined amount of an indeterminate non-A, non-B hepatitis agent. The 60 degrees C, 20-hour treatment method rendered 5.25 logs of the putative acquired immune deficiency syndrome virus, human T-cell lymphotrophic virus III/lymphadenopathy virus, added to factor VIII or factor IX concentrates, undetectable. Heat-treated factor VIII and factor IX complex concentrates prepared by these methods were tested against corresponding untreated control lots. There was no significant difference in the plasma recovery or plasma half-life of the factor (p greater than 0.05). The treated concentrates were equivalent to the control concentrates with respect to vital signs, clinical laboratory studies, and adverse reactions. The heat-treated concentrates appeared bioequivalent to the untreated concentrates with the additional benefit of inactivation of potentially present infectious viruses.

The preoperative treatment of severely anemic patients with a perfluorochemical oxygen-transport fluid, Fluosol-DA.

We gave a perfluorochemical oxygen-transport fluid and plasma expander, Fluosol-DA, to seven severely anemic patients before surgery to determine its effectiveness in supplementing oxygen transport. The dose of Fluosol in the five patients completing the study was 20 ml per kilogram of body weight. When the patients breathed low levels of supplemental oxygen (mean partial pressure of arterial oxygen +/- S.D., 101 +/- 25 torr), the perfluorochemical carried a small amount of oxygen, but when they received pure oxygen (arterial oxygen pressure, 361 +/- 65 torr) it carried approximately 0.8 per cent of oxygen (by volume). This increase accounted for 7 +/- 3 per cent of the patients' arterial oxygen content and 24 +/- 7 per cent of their oxygen consumption. The cardiac index and left ventricular stroke-work index decreased, whereas the oxygen delivery increased, although these changes were not statistically significant. Significant changes included a 22 per cent increase in oxygen consumption, a 59 per cent increase in mixed venous oxygen tension, and an increase in mixed venous hemoglobin saturation to 90 +/- 6 per cent. We conclude that at ambient oxygen tensions fluosol acts primarily as a volume expander, whereas at higher tensions (greater than 300 torr) it contributes substantially to the oxygen-delivery system.

Improvements in the NIOSH registry of toxic effects of chemical substances.

Subsequent to the publication of the 1977 edition of the NIOSH Registry of Toxic Effects of Chemical Substances (RTECS), several additions and changes were introduced that substantially improved the content and accessibility of the RTECS data file. Content additions included primary irritation data for both skin and eyes, in vitro mutagenicity data, and citations to toxicology review articles and to the Toxic Substances Control Act inventory. Also undertaken was a re-evalution of existing tumorigenic entries based on revised selection criteria. Accessibility to RTECS data has been facilitated by the introduction of on-line interactive computer searching, and the preparation and distribution of Computer Output Microfiche (COM-fiche) and magnetic tape copies of the file which supplement the annual publication.

New agents for prostatic cancer activated specifically by prostatic acid phosphatase.

Potential cytotoxic compounds (spindle poisons) are being designed for the treatment of prostatic carcinoma, using the structural requirements of the substrates for prostatic acid phosphatase (PAP). Colchicine has been modified in ring C to give colchiceinamides of substituted ethanolamines and ethanolaminephosphates. Another new series of compounds modifying ring B of thiocolchicine have been prepared. Three O-phosphates of the thiocolchicine series have also been made. One has been examined for its specificity toward PAP. Some toxicity data of these compounds in mice have also been reported. Colchiceinamide-(L)-ephedrinephosphate has been examined in stumptail monkeys and some preliminary results are reported here.

Design of spindle poisons activated specifically by prostatic acid phosphatase (PAP) and new methods for PAP cytochemistry.

By taking advantage of the structural requirements of the substrates for prostatic acid phosphatase (PAP), which consist of steric hindrance and the presence of basic nitrogen in the molecule, potential cytotoxic agents (spindle poisons) are being designed that will become enzyme activated specifically by PAP. Colchicine has been converted to colchiceinamides of substituted ethanolamines and o-phosphoethanolamines. The rate of hydrolysis of the latter by human prostatic tissue as compared to the rate of hydrolysis by human kidneys (P/K ratio) is given and indicates a significant degree of specificity for PAP. Some preliminary toxicity data in mice are also given. New thiocolchicine derivatives with phosphates on ring B are also being prepared for study and some preliminary toxicity data are given. The observation in biochemical experiments that phosphorylcholine is a very specific substrate for PAP has led us to develop specific cytochemical methods for PAP for both light and electron microscopy. Preliminary observations are given and good evidence is provided that PAP is not a lysosomal enzyme, unlike other acid phosphatases. Furthermore, PAP is to other acid phosphatases what the cholinesterases are to other esterases. Since the acid phosphatase that is able to hydrolyze phosphorylcholine is characteristic of prostatic epithelium, this is the acid phosphatase that is referred to be the designation of PAP. Other acid phosphatases (both lysosomal and nonlysosomal) in prostatic epithelial cells are not demonstrated by this substrate and hence are not included in this designation.