Epigallocatechin-3-gallate (EGCG) consumption in the Ts65Dn model of Down syndrome fails to improve behavioral deficits and is detrimental to skeletal phenotypes.

Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability and skeletal deficits. Ts65Dn mice have three copies of ~50% of the genes homologous to Hsa21 and display phenotypes associated with DS, including cognitive deficits and skeletal abnormalities. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including neurological development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have previously shown that EGCG treatment (~10mg/kg/day) improves skeletal abnormalities in Ts65Dn mice, yet the same dose, as well as ~20mg/kg/day did not rescue deficits in the Morris water maze spatial learning task (MWM), novel object recognition (NOR) or balance beam task (BB). In contrast, a recent study reported that an EGCG-containing supplement with a dose of 2-3mg per day (~40-60mg/kg/day) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated if an EGCG dosage similar to that study would yield similar improvements in either cognitive or skeletal deficits. Ts65Dn mice and euploid littermates were given EGCG [0.4mg/mL] or a water control, with treatments yielding average daily intakes of ~50mg/kg/day EGCG, and tested on the multivariate concentric square field (MCSF)-which assesses activity, exploratory behavior, risk assessment, risk taking, and shelter seeking-and NOR, BB, and MWM. EGCG treatment failed to improve cognitive deficits; EGCG also produced several detrimental effects on skeleton in both genotypes. In a refined HPLC-based assay, its first application in Ts65Dn mice, EGCG treatment significantly reduced kinase activity in femora but not in the cerebral cortex, cerebellum, or hippocampus. Counter to expectation, 9-week-old Ts65Dn mice exhibited a decrease in Dyrk1a protein levels in Western blot analysis in the cerebellum. The lack of beneficial therapeutic behavioral effects and potentially detrimental skeletal effects of EGCG found in Ts65Dn mice emphasize the importance of identifying dosages of EGCG that reliably improve DS phenotypes and linking those effects to actions of EGCG (or EGCG-containing supplements) in specific targets in brain and bone.

Differential effects of Epigallocatechin-3-gallate containing supplements on correcting skeletal defects in a Down syndrome mouse model.

SCOPE: Down syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), is characterized by a spectrum of phenotypes including skeletal abnormalities. The Ts65Dn DS mouse model exhibits similar skeletal phenotypes as humans with DS. DYRK1A, a kinase encoded on Hsa21, has been linked to deficiencies in bone homeostasis in DS mice and individuals with DS. Treatment with Epigallocatechin-3-gallate (EGCG), a known inhibitor of Dyrk1a, improves some skeletal abnormalities associated with DS in mice. EGCG supplements are widely available but the effectiveness of different EGCG-containing supplements has not been well studied. METHODS AND RESULTS: Six commercially available supplements containing EGCG were analyzed, and two of these supplements were compared with pure EGCG for their impact on skeletal deficits in a DS mouse model. The results demonstrate differential effects of commercial supplements on correcting skeletal abnormalities in Ts65Dn mice. Different EGCG-containing supplements display differences in degradation, polyphenol content, and effects on trisomic bone. CONCLUSION: This work suggests that the dose of EGCG and composition of EGCG-containing supplements may be important in correcting skeletal deficits associated with DS. Careful analyses of these parameters may lead to a better understanding of how to improve skeletal and other deficits that impair individuals with DS.

Low dose EGCG treatment beginning in adolescence does not improve cognitive impairment in a Down syndrome mouse model.

Down syndrome (DS) or Trisomy 21 causes intellectual disabilities in humans and the Ts65Dn DS mouse model is deficient in learning and memory tasks. DYRK1A is triplicated in DS and Ts65Dn mice. Ts65Dn mice were given up to ~20mg/kg/day epigallocatechin-3-gallate (EGCG), a Dyrk1a inhibitor, or water beginning on postnatal day 24 and continuing for three or seven weeks, and were tested on a series of behavioral and learning tasks, including a novel balance beam test. Ts65Dn as compared to control mice exhibited higher locomotor activity, impaired novel object recognition, impaired balance beam and decreased spatial learning and memory. Neither EGCG treatment improved performance of the Ts65Dn mice on these tasks. Ts65Dn mice had a non-significant increase in Dyrk1a activity in the hippocampus and cerebellum. Given the translational value of the Ts65Dn mouse model, further studies will be needed to identify the EGCG doses (and mechanisms) that may improve cognitive function.

Reactivity of damaged pyrimidines: DNA cleavage via hemiaminal formation at the C4 positions of the saturated thymine of spore photoproduct and dihydrouridine.

Described here are mechanistic details of the chemical reactivities of two modified/saturated pyrimidine residues that represent naturally occurring forms of DNA damage: 5-thyminyl-5,6-dihydrothymine, commonly referred to as the "spore photoproduct" (SP), and 5,6-dihydro-2'-deoxyuridine (dHdU), formed via ionizing radiation damage to cytosine under anoxic conditions and also serving as a general model of saturated pyrimidine residues. It is shown that due to the loss of the pyrimidine C5-C6 double bond and consequent loss of ring aromaticity, the C4 position of both these saturated pyrimidines is prone to the formation of a hemiaminal intermediate via water addition. Water addition is facilitated by basic conditions; however, it also occurs at physiological pH at a slower rate. The hemiaminal species so-formed subsequently converts to a ring-opened hydrolysis product through cleavage of the pyrimidine N3-C4 bond. Further decomposition of this ring-opened product above physiological pH leads to DNA strand break formation. Taken together, these results suggest that once the aromaticity of a pyrimidine residue is lost, the C4 position becomes a "hot spot" for the formation of a tetrahedral intermediate, the decay of which triggers a cascade of elimination reactions that can under certain conditions convert a simple nucleobase modification into a DNA strand break.

Salivary peptide profile and its association with early childhood caries.

BACKGROUND: As dietary management during early childhood is a great barrier in caries control, there is a need for the identification of intrinsic risk factors, capable of allowing the use of a more cost-effective approach to early childhood caries (ECC). OBJECTIVE: To evaluate the salivary peptide profile of children with and without ECC and its association with caries experience. METHODS: One hundred and six 10- to 71-month-old children participated in the study. Caries experience was determined through the visual/tactile method, based on the number of decayed, missing, and filled teeth, and surface scores (dmft/dmfs). Whole saliva was collected for mutans streptococci (MS) detection and peptide analysis. RESULTS: Chromatograms from CF (children without caries experience, n = 58) and CE (children with caries experience, n = 48) saliva pools expressed different patterns. Identification of molecular masses suggested the presence of nine peptides. Three of them were significantly related with caries experience. HNP-3 (α-defensin 3) (P = 0.019) and HBD-3 (ß-defensin 3) (P = 0.034) reduced the chances of experiencing ECC. Proline-rich peptides IB-4 significantly increased caries experience (P = 0.035). Age (P = 0.020) and MS counts (P = 0.036) increased caries experience; however, gender was not associated with dental caries (P = 0.877). CONCLUSION: Specific salivary peptides of CF or CE children in early childhood predispose to a higher or lower risk of caries experience.

Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates.

5-Thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5'-dA radical. The 5'-dA radical is proposed to abstract one of the two H-atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-H(proS) or 6-H(proR) position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-H(proR) atom of SP that is abstracted by the 5'-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis; an H-atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme.

Solid-state and multidimensional solution-state NMR of solid phase extracted and ultrafiltered riverine dissolved organic matter.

In this study we used multidimensional solution-state NMR to elucidate the differences in the chemical composition of solid phase extracted and ultrafiltered DOM isolates. DOM was isolated from water sampled from an oligotrophic river, the River Tagliamento (Italy). The recovery of total DOM was up to 42% with both isolation techniques. In addition to 1- and 2-D solution-state NMR, we also applied 1-D solid-state 13C NMR spectroscopy for DOM characterization. 13C NMR spectroscopy only produced broad overlapping resonances, thus allowing a bulk characterization of DOM composition. However, it demonstrated that the bulk chemical composition of the two DOM fractions exhibited minor spatial-temporal changes. The 2-D experiments (TOCSY, HMQC) showed that the solid phase extracted hydrophobic DOM contained predominantly aliphatic esters, ethers, and hydroxyl groups, whereas the ultrafiltered DOM was comprised partially of peptides/protein, with further evidence for a small amount of aliphatic/fatty acid material. Sugars were present in both DOM fractions. The results show the two isolation techniques selected for different suites of compounds within the bulk DOM pool.

Solid-state carbon-13 nuclear magnetic resonance of humic acids at high magnetic field strengths.

Use of solid-state 13C nuclear magnetic resonance (NMR) spectroscopy has become commonplace in studies of humic substances in soils and sediments, but when modern high-field spectrometers are employed, care must be taken to ensure that the data obtained accurately reflect the chemical composition of these complex materials in environmental systems. In an effort to evaluate the quality of solid-state 13C NMR spectra obtained with modern high-field spectrometers, we conducted a series of experiments to examine spectra of various humic acids taken under a variety of conditions. We evaluate conditions for obtaining semiquantitative cross polarization magic angle spinning (CPMAS) 13C NMR spectra of humic acids at high magnetic field and spinning frequency. We examine the cross polarization (CP) dynamics under both traditional and ramp CP conditions on Cedar Creek humic acid. Fitted equilibrium intensities from these CP dynamic studies compare to within 3.4% of the intensities determined from a Bloch decay spectrum of the same sample. With a 1-ms contact time, ramp CP and traditional CP spectra were acquired on this sample and were found to compare to within 5.4% of the Bloch decay spectrum; however, the signal-to-noise ratio per hour of data acquisition was found to double under ramp CP conditions. These results demonstrate the power of applying modern solid-state NMR techniques at high magnetic field strengths. With these techniques, high-quality, semiquantitative spectra can be quickly produced, allowing the application of solid-state NMR techniques to more environmentally relevant samples, especially those where the quantity is limited.