Nutrition and stem cell integrity in aging.

Adult stem cells (SCs) represent the regenerative capacity of organisms throughout their lifespan. The maintenance of robust SC populations capable of renewing organs and physiological systems is one hallmark of healthy aging. The local environment of SCs, referred to as the niche, includes the nutritional milieu, which is essential to maintain the quantity and quality of SCs available for renewal and regeneration. There is increased recognition that SCs have unique metabolism and conditional nutrient needs compared to fully differentiated cells. However, the contribution of SC nutrition to overall human nutritional requirements is an understudied and underappreciated area of investigation. Nutrient needs vary across the lifespan and are modified by many factors including individual health, disease, physiological states including pregnancy, age, sex, and during recovery from injury. Although current nutrition guidance is generally derived for apparently healthy populations and to prevent nutritional deficiency diseases, there are increased efforts to establish nutrient-based and food-based recommendations based on reducing chronic disease. Understanding the dynamics of SC nutritional needs throughout the life span, including the role of nutrition in extending biological age by blunting biological systems decay, is fundamental to establishing food and nutrient guidance for chronic disease reduction and health maintenance. This review summarizes a 3-day symposium of the Marabou Foundation (www.marabousymposium.org) held to examine the metabolic properties and unique nutritional needs of adult SCs and their role in healthy aging and age-related chronic disease.

Nutrition and its role in human evolution.

Our understanding of human evolution has improved rapidly over recent decades, facilitated by large-scale cataloguing of genomic variability amongst both modern and archaic humans. It seems clear that the evolution of the ancestors of chimpanzees and hominins separated 7-9 million years ago with some migration out of Africa by the earlier hominins; Homo sapiens slowly emerged as climate change resulted in drier, less forested African conditions. The African populations expanded and evolved in many different conditions with slow mutation and selection rates in the human genome, but with much more rapid mutation occurring in mitochondrial DNA. We now have evidence stretching back 300 000 years of humans in their current form, but there are clearly four very different large African language groups that correlate with population DNA differences. Then, about 50 000-100 000 years ago a small subset of modern humans also migrated out of Africa resulting in a persistent signature of more limited genetic diversity amongst non-African populations. Hybridization with archaic hominins occurred around this time such that all non-African modern humans possess some Neanderthal ancestry and Melanesian populations additionally possess some Denisovan ancestry. Human populations both within and outside Africa also adapted to diverse aspects of their local environment including altitude, climate, UV exposure, diet and pathogens, in some cases leaving clear signatures of patterns of genetic variation. Notable examples include haemoglobin changes conferring resistance to malaria, other immune changes and the skin adaptations favouring the synthesis of vitamin D. As humans migrated across Eurasia, further major mitochondrial changes occurred with some interbreeding with ancient hominins and the development of alcohol intolerance. More recently, an ability to retain lactase persistence into adulthood has evolved rapidly under the environmental stimulus of pastoralism with the ability to husband lactating ruminants. Increased amylase copy numbers seem to relate to the availability of starchy foods, whereas the capacity to desaturase and elongate monounsaturated fatty acids in different societies seems to be influenced by whether there is a lack of supply of readily available dietary sources of long-chain polyunsaturated fatty acids. The process of human evolution includes genetic drift and adaptation to local environments, in part through changes in mitochondrial and nuclear DNA. These genetic changes may underlie susceptibilities to some modern human pathologies including folate-responsive neural tube defects, diabetes, other age-related pathologies and mental health disorders.

Emerging concepts on the role of epigenetics in the relationships between nutrition and health.

Understanding the physiological and metabolic underpinnings that confer individual differences in responses to diet and diet-related chronic disease is essential to advance the field of nutrition. This includes elucidating the differences in gene expression that are mediated through programming of the genome through epigenetic chromatin modifications. Epigenetic landscapes are influenced by age, genetics, toxins and other environmental factors, including dietary exposures and nutritional status. Epigenetic modifications influence transcription and genome stability are established during development with life-long consequences. They can be inherited from one generation to the next. The covalent modifications of chromatin, which include methylation and acetylation, on DNA nucleotide bases, histone proteins and RNA are derived from intermediates of one-carbon metabolism and central metabolism. They influence key physiological processes throughout life, and together with inherited DNA primary sequence, contribute to responsiveness to environmental stresses, diet and risk for age-related chronic disease. Revealing diet-epigenetic relationships has the potential to transform nutrition science by increasing our fundamental understanding of: (i) the role of nutrients in biological systems, (ii) the resilience of living organisms in responding to environmental perturbations, and (iii) the development of dietary patterns that programme physiology for life-long health. Epigenetics may also enable the classification of individuals with chronic disease for specific dietary management and/or for efficacious diet-pharmaceutical combination therapies. These new emerging concepts at the interface of nutrition and epigenetics were discussed, and future research needs identified by leading experts at the 26th Marabou Symposium entitled 'Nutrition, Epigenetics, Genetics: Impact on Health and Disease'. For a compilation of the general discussion at the marabou symposium, click here http://www.marabousymposium.org/.

The metabolic role of leucovorin.

Interest in determining if leucovorin, known chemically as 5-formyltetra-hydrofolate, plays a role in one-carbon metabolism is reemerging. While investigations in the 1940s suggested it was an important donor of one-carbon units in folate-mediated biosynthetic reactions, studies between the 1950s and 1980s disproved this hypothesis and dismissed its presence in biological systems as artifactual. Recently, new data has focused attention on the possible biological function of this compound that is widely used in cancer chemotherapy.

Diffraction grade crystals of Escherichia coli serine hydroxymethyltransferase.

Diffraction grade crystals of serine hydroxymethyltransferase were obtained after extensive screening of source of enzyme, purification procedures, ligand binding and crystallization conditions. Serine hydroxymethyltransferase purified from Escherichia coli crystallizes in two forms that are suitable for crystal structure determination.

Murine cysteine dioxygenase gene: structural organization, tissue-specific expression and promoter identification.

The murine gene encoding cysteine dioxygenase (CDO; EC 1.13.11.20), a key enzyme of L-cysteine metabolism, was isolated and characterized, and the proximal promoter was identified. A bacterial artificial chromosome mouse library was screened and a single clone containing the entire CDO gene was isolated. The murine CDO gene contains five exons and spans about 15 kb. The open reading frame is encoded within all five exons. All intron/exon splice junctions and all intron sizes are conserved with the rat CDO gene and are very similar to those of the human CDO gene. The primary transcriptional initiation site is located 213 bp upstream of the initiation ATG codon. The nucleotide sequence of the 5'-promoter region is highly conserved between the mouse and rat genes and contains a TATA-box-like sequence and GC boxes. A variety of consensus cis-acting elements were also identified in the 5'-flanking region. These included HNF-3 beta, HFH-1, HFH-2, HFH-3, C/EBP, and C/EBP beta, all of which are consistent with the tissue-specific expression profiles of the gene. Gene reporter studies of the CDO 5'-region indicated the presence of an active promoter within the first 223 bp upstream of the transcriptional initiation site and the possible presence of repressor elements upstream of bp -223. Northern blot analyses indicated that the CDO gene displays tissue-specific expression, with the highest mRNA level present in liver and with detectable levels found in kidney, lung, brain and small intestine. Western blot analyses indicated that CDO protein levels parallel mRNA levels. These results are consistent with the known function of CDO in whole-body cysteine homeostasis.

Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene.

The human cytoplasmic serine hydroxymethyltransferase (CSHMT) gene was isolated, sequenced and its expression characterized in human MCF-7 mammary carcinoma and SH_5Y5Y neuroblastoma cells. The 23-kb gene contains 12 introns and 13 exons; all splice junctions conform to the gt/ag rule. The open reading frame is interrupted by 10 introns, two of which are positionally conserved within the human mitochondrial SHMT gene. The gene is expressed with 330 nucleotides of 5' untranslated message within three exons. The 5' promoter region does not contain a consensus TATA, and primer extension and 5'-RACE studies suggest that transcription initiation occurs at multiple sites. Consensus motifs for several regulatory proteins, including SP1, mammary and neuronal-specific elements, NF1, a Y-box, and two steroid hormone response elements, are present within the first 408 nucleotides of the 5' promoter region. The human gene is expressed as multiple splice variants in both the 5' untranslated region and within the open reading frame, all due to exon excision. The splicing pattern is cell-specific. At least six CSHMT mRNA splice forms are present in MCF-7 cells; the gene is expressed as a full-length message as well as splice forms that lack exon(s) 2, 9 and 10. In 5Y cells, the predominant form of the message lacks exon 2, which encodes part of the 5' untranslated region, but does not contain deletions within the open reading frame. Western analysis suggests that the CSHMT gene is expressed as a single full-length protein in 5Y cells, but as multiple forms in MCF-7 cells. Multiple tissue Northern blots suggest that the CSHMT message levels and alternative splicing patterns display tissue-specific variations.

First Report of the Pitch Canker Fungus (Fusarium circinatum) in the Sierra Nevada of California.

The pitch canker fungus, Fusarium circinatum (teleomorph Gibberella circinata), was isolated from a branch originating from rootstock of a Douglas-fir (Pseudotsuga menziesii) graft in a breeding orchard at 1,000m elevation in El Dorado County, California. We visited the orchard after the New Zealand Ministry of Agriculture and Forestry reported in November 2003 that a Douglas-fir scion (branch cutting) shipped from there in January-and subsequently grafted and held in a quarantine facility near Christchurch-was infected with the pitch canker fungus. We took samples throughout the orchard of any branches that appeared unhealthy. In addition, asymptomatic branches from the tree alleged to be the source of the New Zealand infestation were collected to assay for propagules of F. circinatum. Wash water from these branches was negative for the pathogen. Likewise, F. circinatum was not recovered from water washings of 20 branches collected randomly throughout the orchard. Fifteen branch samples collected from symptomatic Douglas-fir grafts were cultured on water agar and only one yielded a colony with an appearance consistent with F. circinatum. A single spore subculture of this isolate was confirmed as F. circinatum on the basis of colony morphology and the structure of the microconidiophores (1). The virulence of this isolate was evaluated by inoculating susceptible 2-year-old Monterey pine (Pinus radiata) seedlings with a toothpick to wound the main stem and insert potato dextrose agar colonized by the fungus. Twenty-four days later, pitching and yellow needles were evident at the site of inoculation, and removal of the bark revealed resin-soaked and discolored tissue. Concurrent with the pathogenicity test described above, a culture of the putative F. circinatum isolated in New Zealand was inoculated into Monterey pines with an identical outcome. The fungus was reisolated from lesions from both sets of inoculations and confirmed as F. circinatum based on morphological criteria. Isolates GL285 and GL286 are available from T. R. Gordon upon request. Prior to its discovery in the Sierra Nevada, pitch canker in California was known only from counties on or near the coast. Our report indicates the pathogen can survive and infect trees 110 km east of the previous most-inland site of infestation. It remains to be seen how extensively pitch canker will develop in the Sierra Nevada. Douglas-fir is only moderately susceptible to F. circinatum, which has not been observed to cause significant damage to this species. On the other hand, low-elevation Sierra Nevada pines including P. sabiniana, P. coulteri, and P. ponderosa are substantially more susceptible than are Douglas-fir in greenhouse tests (2). References: (1) T. R. Gordon et al. Mycol. Res. 100:850, 1996. (2) T. R. Gordon et al. Plant Dis. 85:1128, 2001.

Dietary and genetic manipulations of folate metabolism differentially affect neocortical functions in mice.

Converging evidence suggests that folate-mediated one-carbon metabolism may modulate cognitive functioning throughout the lifespan, but few studies have directly tested this hypothesis. This study examined the separate and combined effects of dietary and genetic manipulations of folate metabolism on neocortical functions in mice, modeling a common genetic variant in the MTHFD1 gene in humans. Mutant (Mthfd1(gt/+)) and wildtype (WT) male mice were assigned to a folate sufficient or deficient diet at weaning and continued on these diets throughout testing on a series of visual attention tasks adapted from the 5-choice serial reaction time task. WT mice on a deficient diet exhibited impulsive responding immediately following a change in task parameters that increased demands on attention and impulse control, and on trials following an error. This pattern of findings indicates a heightened affective response to stress and/or an inability to regulate negative emotions. In contrast, Mthfd1(gt/+) mice (regardless of diet) exhibited attentional dysfunction and a blunted affective response to committing an error. The Mthfd1(gt/+) mice also showed significantly decreased expression levels for genes encoding choline dehydrogenase and the alpha 7 nicotinic cholinergic receptor. The effects of the MTHFD1 mutation were less pronounced when combined with a deficient diet, suggesting a compensatory mechanism to the combined genetic and dietary perturbation of folate metabolism. These data demonstrate that common alterations in folate metabolism can produce functionally distinct cognitive and affective changes, and highlight the importance of considering genotype when making dietary folate recommendations.

No association between cSHMT genotypes and the risk of breast cancer in the Nurses' Health Study.

OBJECTIVES: Increased breast cancer risk has been observed with both low folate status and a functional polymorphism in methylenetetrahydrofolate reductase (MTHFR 677C --> T). Cytoplasmic serine hydroxymethyltransferase (cSHMT) affects the flow of one-carbon units through the folate metabolic network, but there is little research on a role for genetic variation in cSHMT in determining breast cancer risk. METHODS: A nested case-control study within the Nurses' Health Study was used to investigate an association between cSHMT (1420C --> T) and breast cancer risk. RESULTS: No evidence for an association between the cSHMT genotype and breast cancer was observed. There was also no evidence of a gene-gene interaction between cSHMT and MTHFR. CONCLUSIONS: There was no evidence of an association between the cSHMT genotype and breast cancer occurrence. Further research in populations with differing average folate intake may be required to fully understand the interactions of folate nutrition, sequence variation in folate genes and breast cancer risk.

5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase.

The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods. These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics. Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex. However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate. The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase. The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism.

Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate.

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.

Evidence for the accumulation of a stable intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate.

Solutions of 5,10-methenyltetrahydropteroylglutamate can be converted to a stable hydrated adduct by heating solutions at 50 degrees C at pH values of 3-5 for several hours. The adduct is stable at pH values from 4 to 9 for hours, but at pH values below 2 it is converted to 5,10-methenyltetrahydropteroylglutamate and at pH values above 8 it is converted to 5-formyltetrahydropteroylglutamate. Arguments are presented that the adduct is (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate formed from (11S)-5,10-hydroxymethylenetetrahydropteroylglutamate by formation of an ylide at C-11 which undergoes inversion of the electron pair to form the (11R) isomer. The (11R) hydrated adducted is believed to be the isomer of 5,10-methenyltetrahydropteroylglutamate referred to as anhydroleucovorin B by Cosulich et al. [Cosulich, D. C., Roth, B., Smith, J. M., Hultquist, M. E., & Parker, R. P. (1952) J. Am. Chem. Soc. 74, 3252-3263]. In addition, a new mechanism for the formation of 5-formyltetrahydropteroylglutamate from either 5,10-methenyltetrahydropteroylglutamate or 10-formyltetrahydropteroylglutamate via (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate is proposed. A requirement for this pathway is that the formyl proton of 10-formyltetrahydropteroylglutamate exchange with solvent protons. The exchange of this formyl proton was observed at all pH values from 5.5 to 11.5 at a rate which exceeded by more than an order of magnitude the rate of formation of 5-formyltetrahydropteroylglutamate.

Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.

Serine hydroxymethyltransferase in the presence of glycine catalyzes the hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamate to (6S)-5-formyltetrahydropteroylpolyglutamate. The enzyme also catalyzes the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from a compound in equilibrium with (6R)-5,10-methenyltetrahydropteroylpolyglutamate believed to be (6R,11R)-5,10-hydroxymethylenetetrahydropteroylpolyglutamate , a putative intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate [Stover, P., & Schirch, V. (1992) Biochemistry (preceding paper in this issue)]. The enzymatic mechanism for the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from these substrates and the role of glycine in the reaction was addressed. Evidence suggests that (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is a catalytically competent intermediate in the enzyme-catalyzed hydrolysis of (6R)-5,10-methenyltetrahydropteroyltetraglutamate. The enzyme displays a high Km of 40 microM for (6R)-5,10-methenyltetrahydropteroyltetraglutamate, while the Km for (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is below 0.5 microM. The kcat values for both reactions are identical and equal to the rate of formation of an enzyme ternary complex absorbing at 502 nm which is formed from glycine and (6S)-5-formyltetrahydropteroylpolyglutamate. The hydrolysis reaction proceeds with exchange of the C11 formyl proton of (6R)-5,10-methenyltetrahydropteroyltetraglutamate, suggesting that the enzyme-catalyzed reaction occurs by the same C11 carbanion inversion mechanism as the nonenzymatic reaction. Isotope exchange experiments using [2-3H]glycine and differential scanning calorimetry data suggest both a catalytic and a conformational role for glycine in the enzymatic reaction. The results are discussed in terms of the similarity in mechanisms of the SHMT-catalyzed retroaldol cleavage of serine and hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamates.

Synthesis of (6S)-5-formyltetrahydropteroyl-polyglutamates and interconversion to other reduced pteroylpolyglutamate derivatives.

5-Formyltetrahydropteroylpolyglutamates can be synthesized and purified directly from dihydropteroylpolyglutamates in a single-step procedure without purification of intermediates and with yields greater than 90%. The procedure involves a coupled enzymatic synthesis of 10-formyltetrahydropteroylpolyglutamates using the enzymes dihydrofolate reductase, serine hydroxymethyltransferase, and C1-tetrahydrofolate synthase with catalytic amounts of NADPH. The 10-formyltetrahydropteroylpolyglutamates are subsequently converted to 5-formyltetrahydropteroylpolyglutamates at 90 degrees C with near quantitative yields. 5-Formyltetrahydropteroylpolyglutamates are the only stable reduced derivatives of tetrahydropteroylpolyglutamates and can be purified and stored indefinitely without decomposition. Additionally, 5-formyltetrahydropteroylpolyglutamates can be readily converted to other derivatives of tetrahydropteroylpolyglutamates with yields greater than 95%. Also described is the synthesis of tetrahydropteroylglutamates labeled at C-11 with either 14C or 13C. Rapid purification procedures for serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from frozen rabbit livers are presented.

A survey of polar and nonpolar lipids from epidermis and epidermal appendages of the chicken (Gallus domesticus).

Total lipid was extracted from chicken (Gallus domesticus) epidermis, leg scale, claws, feathers and preen glands and analyzed by quantitative thin-layer chromatography. All of the tissue lipids contained large proportions of wax diesters, triglycerides, and free sterols and variable proportions of phospholipids, steryl esters and free fatty acids. All of the keratinized tissues, but not the preen gland, contained ceramides, acylceramides and cholesteryl sulfate. Acylglucosylceramides were found only in full thickness epidermis. Glucosylsterols and acylglucosylsterols were found in the keratinized tissues, and may be of significance in the evolutionary history of the epidermal water barrier.

New perspectives on folate catabolism.

Folate catabolism has been assumed to result from the nonenzymatic oxidative degradation of labile folate cofactors. Increased rates of folate catabolism and simultaneous folate deficiency occur in several physiological states, including pregnancy, cancer, and when anticonvulsant drugs are used. These studies have introduced the possibility that folate catabolism may be a regulated cellular process that influences intracellular folate concentrations. Recent studies have demonstrated that the iron storage protein ferritin can catabolize folate in vitro and in vivo, and increased heavy-chain ferritin synthesis decreases intracellular folate concentrations independent of exogenous folate levels in cell culture models. Ferritin levels are elevated in most physiological states associated with increased folate catabolism. Therefore, folate catabolism is emerging as an important component in the regulation of intracellular folate concentrations and whole-body folate status.