Neural stem cells reduce hippocampal tau and reelin accumulation in aged Ts65Dn Down syndrome mice.

Tau accumulation, in the form of neurofibrillary tangles (NFT), is an early neuropathological characteristic of Alzheimer's disease (AD) and early onset AD frequently seen in Down syndrome (DS). We investigated the presence of tau accumulation in the brains of aging DS mice using the Ts65Dn mouse model. All aged mice appeared to have substantial clusters of extracellular granules that were positive for tau and reelin, but not for amyloid-ß or APP. These clusters were found primarily in CA1 of the hippocampus. In addition, the aged trisomic DS mice had a significantly greater accumulation of extracellular tau/reelin granular deposits compared to disomic littermates. These granules were similar to those described by others who also found extracellular proteinous granules in the brains of non-DS mice engineered to model aging and/or AD. When neural stem cells (NSC) were implanted unilaterally into the hippocampus of the Ts65Dn mice, the tau/reelin-positive granules were significantly reduced in both trisomic and disomic mice. Our findings indicate that changes in tau/reelin-positive granules could be used as an index for neuropathological assessment in aging DS and AD. Furthermore, changes in granule density could be used to test the efficacy of novel treatments, such as NSC implantation. Lastly, it is speculated that the unique abilities of NSC to migrate and express growth factors might be a contributing factor to reducing tau/reelin accumulation in aging DS and AD.

Betaine supplementation improves the atherogenic risk factor profile in a transgenic mouse model of hyperhomocysteinemia.

OBJECTIVE: To investigate the lipotropic action of betaine on plasma lipoproteins and tissue lipids. METHODS AND RESULTS: Adult mice, wild type (+/+) or heterozygous (+/-) for a disruption of the methylenetetrahydrofolate reductase (Mthfr) gene, were supplemented with betaine for 1 year and compared with mice on control diets. Outcome measures were plasma homocysteine and lipoprotein levels, aortic and liver morphology, and liver staining for 3-nitrotyrosine (oxidative stress marker) and Apolipoprotein A-I (ApoA-I). We also investigated short-term effects of supplemental betaine on plasma lipoproteins in Mthfr +/+ and +/- mice. Both genotypes showed significantly lower plasma homocysteine after long-term betaine supplementation, and lower plasma triglycerides and higher HDL-cholesterol after both short- and long-term betaine. Lipid accumulation in liver and aortic wall tended to be lower in Mthfr+/+ compared to Mthfr+/- mice and in betaine-supplemented compared to unsupplemented mice. Nitrotyrosine staining was higher and ApoA-I staining was lower in livers of Mthfr+/- compared to Mthfr+/+ mice. Betaine did not affect staining of nitrotyrosine but increased ApoA-I staining. A significant negative correlation was observed between plasma homocysteine and liver ApoA-I. CONCLUSIONS: Mild MTHFR deficiency in mice is associated with increased risk for atherosclerotic disease. Betaine has a lipotropic effect, which is associated with a reduction in homocysteine, an increase in ApoA-I and an amelioration of the atherogenic risk profile.

Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region.

Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.

Transsulfuration in Saccharomyces cerevisiae is not dependent on heme: purification and characterization of recombinant yeast cystathionine beta-synthase.

Cystathionine beta-synthase [CBS; L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration in both yeast and humans. It has been established previously that human CBS is a hemeprotein but although the heme group appears to be essential for CBS activity, the exact function of the heme group is unknown. CBS activity is absent in heme deficient strains of Saccharomyces cerevisiae grown without heme supplementation. CBS activity can be restored by supplementing these strains with heme, implying that there is a heme requirement for yeast CBS. We subcloned, overexpressed and purified yeast CBS. The yeast enzyme shows absolute pyridoxal 5'-phosphate (PLP) dependence for activity but we could find no evidence for the presence of a heme group. Given the degree of sequence and mechanistic similarity between yeast and human CBS, this result indicates that heme is unlikely to play a direct catalytic role in the human CBS reaction mechanism. Further characterization revealed that, in contrast to human CBS, S-adenosylmethionine (AdoMet) does not activate yeast CBS. Yeast CBS was found to be coordinately regulated with proliferation in S. cerevisiae. This finding is the most likely explanation of the observed apparent heme dependence of transsulfuration in vivo.

The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms.

Cystathionine beta-synthase [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.

Differential effects of sodium butyrate on the transcription of the human TIS11 family of early-response genes in colorectal cancer cells.

The tetradecanoyl phorbol acetate (TPA)-inducible sequence 11 (TIS11) family of early-response proteins consists of at least five members. They share a highly conserved Cys3His zinc-binding motif, but otherwise have little sequence similarity. Their function remains unknown, but all are induced rapidly and transiently in response to extracellular hormone and growth factor signals. Sodium butyrate, a fermentation product of dietary fibre, effects colorectal cancer cell proliferation by inducing growth arrest, differentiation and apoptosis. In this communication, we report that butyrate has differential effects on the transcription of the three human TIS11 family members identified so far in T84 and HT-29 human colorectal cancer cell lines. Butyrate response factor 1 (BRF1) transcription is repressed, butyrate response factor 2 (BRF2) transcription is activated and there is no apparent effect on the transcription of human TIS11 (HTIS11). Induction and repression occur rapidly, with altered mRNA levels detectable within 15 min of butyrate addition. Two other short-chain fatty acids, propionate and acetate, have no detectable effects on BRF1 or BRF2 transcription.

The human immediate early gene BRF1 maps to chromosome 14q22-q24.

BRF1 (Butyrate response factor 1) is a member of an immediate early gene family specifying putative nuclear transcription factors. A repeat motif incorporating two Cys and two His is highly conserved between family members identified from yeast, Drosophila, mouse, rat, and human. The chromosome localization of none of the human genes has been determined thus far. Using the polymerase chain reaction on a human-rodent hybrid panel, we have localized BRF1 to chromosome 14. This was confirmed by direct sequencing of the PCR fragment. Using fluorescence in situ hybridization, the chromosome localization of BRF1 was further determined as 14q22-q24.

ERF-2, the human homologue of the murine Tis11d early response gene.

A human cDNA specifying a member of the Tis11 early response gene family was cloned and sequenced. The human gene differs from its mouse homologue by encoding an additional 97 amino acids at its C-terminal end. The sequence has transactivation-like motifs, an unusual Cys-Ser-Ala-rich motif and displays sequence similarity at the extreme C-terminal end with another Tis11 family member, ERF-1.