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Pyrroloquinoline quinone (PQQ) is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. In addition, PQQ attenuates clinically relevant dysfunctions (e.g., those associated with ischemia, inflammation and lipotoxicity). PQQ is novel among biofactors that are not currently accepted as vitamins or conditional vitamins. For example, the absence of PQQ in diets produces a response like a vitamin-related deficiency with recovery upon PQQ repletion in a dose-dependent manner. Moreover, potential health benefits, such as improved metabolic flexibility and immuno-and neuroprotection, are associated with PQQ supplementation. Here, we address PQQ’s role as an enzymatic cofactor or accessory factor and highlight mechanisms underlying PQQ’s actions. We review both large scale and targeted datasets demonstrating that a neonatal or perinatal PQQ deficiency reduces mitochondria content and mitochondrial-related gene expression. Data are reviewed that suggest PQQ’s modulation of lactate acid and perhaps other dehydrogenases enhance NAD+-dependent sirtuin activity, along with the sirtuin targets, such as PGC-1α, NRF-1, NRF-2 and TFAM; thus, mediating mitochondrial functions. Taken together, current observations suggest vitamin-like PQQ has strong potential as a potent therapeutic nutraceutical.

Antioxidant supplementation can reverse hepatic steatosis, suggesting dietary antioxidants might have potential as therapeutics for nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). An extraordinarily potent dietary antioxidant is pyrroloquinoline quinone (PQQ). PQQ is a ubiquitous, natural, and essential bacterial cofactor found in soil, plants, and interstellar dust. The major source of PQQ in mammals is dietary; it is common in leafy vegetables, fruits, and legumes, especially soy, and is found in high concentrations in human and mouse breast milk. This chapter reviews chemical and biological properties enabling PQQ's pleiotropic actions, which include modulating multiple signaling pathways directly (NF-κB, JNK, JAK-STAT) and indirectly (Wnt, Notch, Hedgehog, Akt) to improve liver pathophysiology. The role of PQQ in the microbiome is discussed, as PQQ-secreting probiotics ameliorate oxidative stress–induced injury systemwide. A limited number of human trials are summarized, showing safety and efficacy of PQQ.

For nutrition and its associated disciplines, ethical considerations related to research are often complicated by factors that range from the use of experimental research designs that are overly holistic to inextricable links between nutrition research and marketing. As a consequence, there is the need for constant vigilance to assess and deal with apparent conflicts of interest. Also, there are few scientific disciplines that are defined by cultural, religious, or political codifications as is nutrition. Accordingly, examples of historical, cultural, and political events are described that have influenced ethical approaches related to nutrition research. Furthermore, nutrition research questions are often multifaceted and require dealing with complex variables. In this regard, ethical principles and perspectives that have relevance to data acquisition, the publication and translation of nutrition research, and the marketing of nutritional products and concepts are highlighted.

PQQ (pyrroloquinoline quinone) improves energy utilization and reproductive performance when added to rodent diets devoid of PQQ. In the present paper we describe changes in gene expression patterns and transcriptional networks that respond to dietary PQQ restriction or pharmacological administration. Rats were fed diets either deficient in PQQ (PQQ−) or supplemented with PQQ (approx. 6 nmol of PQQ/g of food; PQQ+). In addition, groups of rats were either repleted by administering PQQ to PQQ− rats (1.5 mg of PQQ intraperitoneal/kg of body weight at 12 h intervals for 36 h; PQQ−/+) or partially depleted by feeding the PQQ− diet to PQQ+ rats for 48 h (PQQ+/−). RNA extracted from liver and a Codelink® UniSet Rat I Bioarray system were used to assess gene transcript expression. Of the approx. 10000 rat sequences and control probes analysed, 238 were altered at the P

The expression of phase I and II biotransformation enzymes was examined with respect to experimental diet composition and with the addition of the bi-functional inducer flavone. Enzymatic activity and mRNA levels of cytochrome P450 monooxygenase (CYP) isoforms (CYP1A1, CYP1A2, CYP2B1/2) and glutathione-S-transferase (GST) isoforms (GSTA, GSTM, and GSTP) were used as indices for the changes in expression. An amino acid based (AA) diet and a semi-purified egg white (EW) diet were designed to include similar levels of nutrients and were compared to a standard laboratory chow (SC) diet. Rats (Sprague-Dawley) and mice (C57BL/6) were used as animal models. Animals were fed one of the three diets for 7 days prior to incorporation of flavone (2%, wt/wt). Diets with or without flavone were next fed for an additional 3 days. Enzymatic activities of the CYPs in mice and GSTs in both mice and rats were determined. In mice, the relative mRNA levels for each of the CYP and GST isoforms were also measured. The increase in phase I and II enzyme expression observed in response to flavone was most dynamic when the AA-based diet was used (often >20-fold for given isoform enzymatic activities and >200-fold for specific mRNAs), followed by the EW diet (10 to 20-fold and 100 to 200-fold, respectively). The SC diet resulted in a higher level of background expression of CYP and GST isoforms and as a consequence the observed fold increases in CYP and GST isoforms (enzymatic and mRNA levels) were substantially less (1 to 10-fold and 1 to 150-fold. respectively), when the SC diet fed group with or without flavone was compared.

Cofactors include an array of compounds that are used to improve the catalytic efficiency of biological reactions, particularly those catalyzed by enzymes. Cofactors are derived from vitamins and essential minerals or made in situ . Cofactors serve principally as intermediates in transfer reactions or as cosubstrates. One or more cofactors may be associated with the enzyme's catalytic site(s). Regulation of cofactors involves (1) specific transporters to control cofactor concentrations within cells, (2) chemical modifications to enhance function, and/or (3) linkage between cofactor production and the metabolic processes in which they serve as catalysts.

Sirtuin (Sirt) 1 and Sirt 3 are nicotinamide adenine dinucleotide ((+))-dependent protein deacetylases that are important to a number of mitochondrial-related functions; thus, identification of sirtuin activators is important. Herein, we hypothesize that pyrroloquinoline quinone (PQQ) can act as a Sirt1/Sirt3 activator. In HepG2 cell cultures, PQQ increased the expression of Sirt1 and Sirt3 gene, protein, and activity levels (P < .05). We also observed a significant increase in nicotinamide phosphoribosyltransferase gene expression (as early as 18 hours) and increased NAD(+) activity at 24 hours. In addition, targets of Sirt1 and Sirt3 (peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1 and 2, and mitochondrial transcription factor A) were increased at 48 hours. This is the first report that demonstrates PQQ as an activator of Sirt1 and Sirt3 expression and activity, making it an attractive therapeutic agent for the treatment of metabolic diseases and for healthy aging. Based on our study and the available data in vivo, PQQ has the potential to serve as a therapeutic nutraceutical, when enhancing mitochondrial function.

When pyrroloquinoline quinone (PQQ) is added to an amino acid-based, but otherwise nutritionally complete basal diet, it improves growth-related variables in young mice. We examined PQQ and mitochondrial function based on observations that PQQ deficiency results in elevated plasma glucose concentrations in young mice, and PQQ addition stimulates mitochondrial complex 1 activity in vitro. PQQ-deficient weanling mice had a 20-30% reduction in the relative amount of mitochondria in liver; lower respiratory control ratios, and lower respiratory quotients than PQQ-supplemented mice (2 mg PQQ/kg diet). In mice from dams fed a conventional laboratory diet, but switched at weaning to the basal diet, plasma glucose, Ala, Gly, and Ser concentrations were elevated at 4 wk (PQQ- vs. PQQ+), but not at 8 wk. The relative mitochondrial content (ratio of mtDNA to nuclear DNA) also tended (P

Copper-deficient rat embryos are characterized by brain and heart anomalies, low superoxide dismutase activity, and high superoxide anion concentrations. One consequence of increased superoxide anions can be the formation of peroxynitrite, a strong biological oxidant. To investigate developmentally important features of copper deficiency, GD 8.5 mouse embryos from copper-adequate and copper-deficient dams were cultured in media that were adequate or deficient in copper. After 48 h, copper-deficient embryos exhibited brain and heart anomalies, and a high incidence of yolk sac vasculature abnormalities compared to controls. Immunohistochemistry of 4-hydroxynonenal and 8-hydroxy-2′-deoxyguanosine for lipid and DNA damage, respectively, was similar between groups. In contrast, 3-nitrotyrosine, taken as a measure of protein nitration, was markedly higher in the neuroepithelium of the anterior neural tube of copper-deficient embryos than in controls. Repletion of copper-deficient media with copper, or supplementation with copper-zinc superoxide dismutase, Tiron, or glutathione peroxidase did not ameliorate the abnormal development, but did decrease 3-nitrotyrosine in neuroepithelium of copper-deficient embryos. These data support the concept that while copper deficiency compromises oxidant defense and increases protein nitration, additional mechanisms, e.g., altered nitric oxide metabolism may contribute to copper-deficiency-induced teratogenesis.

Perturbations in copper (Cu) metabolism are a characteristic of diabetes, for example, elevated plasma Cu and compromised oxidant defense related to diabetes-induced effects on Cu-containing enzymes. Herein, the redistribution of Cu in selected tissues is described in response to diabetic and nondiabetic states in rats that were fed diets adequate in (12 mg Cu/kg of diet) or deficient in (no added Cu) Cu. Diabetes was induced by intravenous administration of streptozotocin (40 mg/kg body weight). After 5 weeks, rats were gavaged with (67)Cu (0.74 MBq per rat) using the Cu-deficient diet as a vehicle (suspended 1:3 in water) and killed at various time points. The use of (67)Cu allowed for the assessment of short-term Cu distribution and its comparison to the steady-state Cu distribution, as determined by direct Cu analysis. In contrast to control rats, the adaptive mechanisms for Cu homeostasis in diabetic rats were impaired. In general, measures of Cu retention were reduced in diabetic rats compared to corresponding values for control rats. Moreover, diabetic rats had low copper, zinc superoxide dismutase activity that was reduced even further when diabetic rats were fed with low-Cu diets. However, liver and kidney metallothionein and plasma ceruloplasmin levels were elevated in diabetic rats compared to control rats. Such diabetes-related metabolic alterations were taken as measures of increased oxidative stress and inflammation, which may have implications in the progression of diabetes-related pathologies.

The workshop and exhibits dealing with analytical methods were selected to highlight the current state of the art in elemental analysis. The presentations in the first part of the workshop described approaches and advances important to the analysis of trace minerals. These presentations included: 1) two approaches to mass spectrometry, inductively coupled plasma and accelerator mass spectrometry; 2) use of nuclear magnetic resonance in studies of mineral function; and 3) the use and limitations of fluorescent probes in studies of metal uptake and regulation. In the second part of the workshop, the International Atomic Energy's contributions to nutritional "metrology" were described. Advances in instrumentation over the past decade have led to extraordinary improvements in the precision and sensitivity of mineral analyses. The ability to address isotopic speciation at such low levels sets the stage for numerous novel approaches in the assessment of trace mineral function.

The teratogenicity of copper (Cu) deficiency may result from increased oxidative stress and oxidative damage. Dams were fed either control (8.0 μg Cu/g) or Cu-deficient (0.5 μg Cu/g) diets. Embryos were collected on Gestational Day 12 for in vivo studies or on Gestational Day 10 and cultured for 48 h in Cu-deficient or Cu-adequate media for in vitro studies. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GR) activities were measured in control and Cu-deficient embryos as markers of the oxidant defense system. Superoxide anions were measured as an index of exposure to reactive oxygen species (ROS). No differences were found in GPX or GR activities among treatment groups. However, SOD activity was lower and superoxide anion concentrations higher in Cu-deficient embryos cultured in Cu-deficient serum compared to control embryos cultured in control serum. Even so, Cu-deficient embryos had similar CuZnSOD protein levels as controls. In the in vitro system, Cu-defi...

The dietary requirements for most water-soluble vitamins in homeothermic animals, particularly vitamins utilized in energy-related pathways, are related directly to metabolic rate. As a consequence, vitamin requirements are similar when expressed relative to empirical functions of metabolic body size, e.g. (Wtkg)3/4 or body surface area. The vitamin requirements for a range of animals are expressed relative to their corresponding rates of basal metabolism. Data for the rates of ascorbic acid production and turnover in animals that produce ascorbic acid are also compared with the ascorbic acid requirements and turnover in humans and guinea pigs, species that require ascorbic acid as a dietary essential. Factors that are most important in dictating the relative need for a given vitamin from a chemical perspective include chemical stability, the relative number of catalytic events that are involved in the process, the nature of the interactions with associated enzymes, and the presence or absence of pathways for partial synthesis or regeneration of the given vitamin. Vitamins that are required daily in millimolar amounts are usually less stable chemically, are involved in numerous reactions, and often exist in tissues as dissociable cofactors. In contrast, vitamins that are required daily in micromolar amounts are involved in fewer reactions and are often covalently bound to the proteins or enzymes for which they serve as cofactors. Development of the preceding concepts allows linkages of relative vitamin requirements to energy utilization and related biochemical and oxidative processes, which can aid in developing a better understanding of the integrative nature of nutritional and biochemical relationships.

The observation of accelerated collagen glycation in association with enhanced progression of many age-associated diseases in hyperglycemic subjects has led researchers to propose a role of glycation in the aging process. Although short-term studies in healthy animals suggest that feeding a diet high in fructose may increase serum glucose concentrations and increase glycemic stress, the effects of a long-term feeding, i.e., life span, are unknown. This study was designed to evaluate the long-term effects of dietary carbohydrates on serum and tissue markers of glycemic stress. Three-month-old male Fischer 344 rats were given free access to or restricted to 60% caloric intake of one of five isocaloric diets that contained as their carbohydrate source either cornstarch, glucose, sucrose, fructose or equimolar amounts of fructose and glucose. Rats were killed at 9-, 18- or 26-mo of age. Glycated hemoglobin, serum glucose and fructosamine levels were measured as markers of serum glycemic stress. Collagen-associated fluorescence and pentosidine concentrations were measured in skin, aortic, tracheal and tail tendon collagen as markers of advanced glycation endproducts (AGE). The source of dietary carbohydrate had little effect on markers of glycemic stress and the accumulation of AGE. Restricting the amount of calories consumed resulted in lower serum glucose concentrations, glycated hemoglobin levels and pentosidine concentrations in tail tendon collagen. Our data suggest that the rate of collagen glycation is tissue-specific. These results suggest that long-term feeding of specific dietary carbohydrates does not alter serum glucose concentrations or the rate of collagen glycation. Rather, age-related accumulation of AGE is more closely related to caloric intake.

O-quinone cofactors derived from tyrosine and tryptophan are involved in novel biological reactions that range from oxidative deaminations to free-radical redox reactions. The formation of each of these cofactors appears to involve post-translational modifications of either tyrosine or tryptophan residues. The modifications result in cofactors, such as topaquinone (TPQ), tryptophan tryptophylquinone (TTQ), lysine tyrosylquinone (LTQ) or the copper-complexed cysteinyl-tyrosyl radical from metal-catalyzed reactions. Pyrroloquinoline quinone (PQQ) appears to be formed from the annulation of peptidyl glutamic acid and tyrosine residues stemming from their modification as components of a precursor peptide substrate. PQQ, a primary focus of this review, has invoked considerable interest because of its presence in foods, antioxidant properties and role as a growth-promoting factor. Although no enzymes in animals have been identified that exclusively utilize PQQ, oral supplementation of PQQ in nanomolar amounts increases the responsiveness of B- and T-cells to mitogens and improves neurologic function and reproductive outcome in rodents. Regarding TPQ and LTQ, a case may be made that the formation of TPQ and LTQ is also influenced by nutritional status, specifically dietary copper. For at least one of the amine oxidases, lysyl oxidase, enzymatic activity correlates directly with copper intake. TPQ and LTQ are generated following the incorporation of copper by a process that involves the two-step oxidation of a specified tyrosyl residue to first peptidyl dopa and then peptidyl topaquinone to generate active enzymes, generally classed as "quinoenzymes." Limited attention is also paid to TTQ and the copper-complexed cysteinyl-tyrosyl radical, cofactors important to fungal and bacterial redox processes.

Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.

Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.

Rat embryos (gestation days 9.0 and 10.0) obtained from dams that were fed a Cu-adequate (8 micrograms Cu/g) or Cu-deficient (< 0.5 micrograms Cu/g diet were cultured for 48 hr in Cu-adequate (16.2 microM) or Cu-deficient (1.0 microM) rat serum. Control embryos cultured in control serum were morphologically normal. Embryos from Cu-deficient dams developed abnormally when cultured in Cu-deficient serum; the abnormalities included distended hindbrains, blisters, blood pooling, and cardiac defects. Control embryos cultured in Cu-deficient serum and Cu-deficient embryos cultured in control serum also showed abnormal development, but to a lesser degree than that of the Cu-deficient embryos cultured in Cu-deficient serum. To test the idea that the above abnormalities were due in part to free radical induced damage occurring secondary to an impaired oxidant defense system, a chemiluminescence assay was used to detect superoxide dismutase (SOD) activity in the cultured embryos. SOD activity was lowest in embryos cultured in Cu-deficient serum. When the Cu-deficient serum was supplemented with antioxidants (CuZnSOD or glutathione peroxidase), its teratogenicity was reduced. These data support the idea that an impaired oxidant defense system contributes to the dysmorphology associated with Cu deficiency. However, the Cu-deficient embryos also had low cytochrome c oxidase activity compared to control embryos--thus, multiple factors are likely contributing to Cu deficiency-induced abnormalities.

Copper deficiency during embryonic and fetal development can result in numerous gross structural and biochemical abnormalities. Such a deficiency can arise through a variety of mechanisms, including low maternal dietary copper intake, disease-induced or drug-induced changes in maternal and conceptus copper metabolism, or both. These issues are discussed in this article along with the use of in vitro embryo culture models to study the mechanisms underlying copper deficiency-induced teratogenesis. Current data suggest that changes in free radical defense mechanisms, connective tissue metabolism, and energy production can all contribute to the dysmorphogenesis associated with developmental copper deficiency.

The functions of vitamins and selected vitamin-like biofactors are summarized. Basic mechanisms of actions are described that are important to an understanding of the deficiency signs or symptoms that are observed when such compounds are limiting in the diet of animals. With respect to given biofactors, primary and secondary metabolites that are present in diets, compounds with definable health-related functions are highlighted. Particular attention is given to secondary metabolites that influence oxidative metabolism and mitochondrial function.

Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation.

The essentiality of zinc for normal brain development is well established. It has been suggested that primary and secondary zinc deficiencies can contribute to the occurrence of numerous human birth defects, including many involving the central nervous system. In a recent study, we searched for zinc transporter genes that were critical for neurodevelopment. We confirmed that ZIP12 is a zinc transporter encoded by the gene slc39a12 that is highly expressed in the central nervous systems of human, mouse, and frog (Xenopus tropicalis).Using loss-of-function methods, we determined that ZIP12 is required for neuronal differentiation and neurite outgrowth and necessary for neurulation and embryonic viability. These results highlight an essential need for zinc regulation during embryogenesis and nervous system development. We suggest that slc39a12 is a candidate gene for inherited neurodevelopmental defects in humans.

Zn 2+ is required for many aspects of neuronal structure and function. However, the regulation of Zn 2+ in the nervous system remains poorly understood. Systematic analysis of tissue-profiling microarray data showed that the zinc transporter ZIP12 ( slc39a12 ) is highly expressed in the human brain. In the work reported here, we confirmed that ZIP12 is a Zn 2+ uptake transporter with a conserved pattern of high expression in the mouse and Xenopus nervous system. Mouse neurons and Neuro-2a cells produce fewer and shorter neurites after ZIP12 knockdown without affecting cell viability. Zn 2+ chelation or loading in cells to alter Zn 2+ availability respectively mimicked or reduced the effects of ZIP12 knockdown on neurite outgrowth. ZIP12 knockdown reduces cAMP response element-binding protein activation and phosphorylation at serine 133, which is a critical pathway for neuronal differentiation. Constitutive cAMP response element-binding protein activation restores impairments in neurite outgrowth caused by Zn 2+ chelation or ZIP12 knockdown. ZIP12 knockdown also reduces tubulin polymerization and increases sensitivity to nocodazole following neurite outgrowth. We find that ZIP12 is expressed during neurulation and early nervous system development in Xenopus tropicalis, where ZIP12 antisense morpholino knockdown impairs neural tube closure and arrests development during neurulation with concomitant reduction in tubulin polymerization in the neural plate. This study identifies a Zn 2+ transporter that is specifically required for nervous system development and provides tangible links between Zn 2+ , neurulation, and neuronal differentiation.

Lysyl oxidase levels were estimated in rat tissues using an enzyme-linked immunosorption assay (ELISA) and a functional assay standardized against known amounts of purified lysyl oxidase. High concentrations of lysyl oxidase (or = 150 micrograms/g of tissue or packed cells) were detected in connective tissues, such as tendon and skin. Values for aorta, kidney, lung and liver ranged from 30 to 150 micrograms/g of tissue; values for skeletal muscle and diaphragm were30 micrograms/g tissue. Purified rat skin lysyl oxidase catalyzed the release of 50-100 Bq of tritium per micrograms enzyme in assays that used 3H-elastin-rich substrates. In dense connective tissues, good agreement was obtained for the values from ELISA and those derived from measurements of functional activity in aorta, lung, skin and tendon (r20.9). When egg white-based experimental diets containing 2 or 10 micrograms/g added copper were fed to weanling rats, values for skin lysyl oxidase functional activity in the group fed 2 micrograms/g added copper were one-third to one-half the values for skin lysyl oxidase functional activity in rats fed 10 micrograms/g copper. This reduction in lysyl oxidase activity, however, had minimal effect on indices of collagen maturation in rat skin, e.g., collagen solubility in neutral salt and dilute acid or the levels of acid stable cross-links. Moreover, copper deficiency did not influence the steady-state levels of lysyl oxidase specific mRNA in rat skin or the apparent amounts of lysyl oxidase in rat skin as determined by ELISA. These observations underscore that the concentration of lysyl oxidase is relatively high in dense corrective tissues, and although decreasing dietary copper influences functional activity, there is little apparent effect on the production of lysyl oxidase protein.

The effect of a metallothionein (MT) antisense oligodeoxynucleotide (ODN) on mouse preimplantation embryo development was investigated. Preimplantation embryos were cultured for 72 h and examined following exposure to either an MT antisense or sense ODN. Blastocyst formation (cavitation) and embryo cell number were lower in embryos exposed to the MT antisense ODN than in controls or in embryos exposed to the MT sense ODN. In embryos cultured in medium containing free nucleotides, cavitation frequency was not affected, although mean embryo cell number was lower than in controls. Combined, this work shows that an antisense ODN against MT can significantly affect blastocyst formation of preimplantation embryos; some, but not all, of the observed effects on embryo cell number may have been due to nucleotide toxicity.

This study was designed to investigate the effects of aging and caloric restriction on collagen deposition in a model of wound healing. Polyvinyl alcohol sponges were implanted in Fischer 344 rats aged 6, 10, or 24 months. Sponges were removed after 7, 10, or 14 days and analyzed for hydroxyproline content and for enzymatic crosslinks. Aging was associated with a decrease in collagen accumulation as well as a delay in reaching peak rates of collagen accumulation. Levels of enzymatic crosslinks were increased in granulation tissue from old animals relative to young animals. Caloric restriction (60% of ad libitum intake) maintained only during the 2-week experimental period did not affect collagen accumulation, but did result in decreased levels of the difunctional crosslink dihydroxylysinonor-leucine (DHLNL) in sponges implanted for 10 days, but not 14 days. Caloric restriction initiated 4 months before sponges were implanted resulted in decreased collagen accumulation and decreases in DHLNL at all implantation times. These studies suggest that: (1) aging affects total collagen production as well as rate of collagen production in response to injury; (2) aging is not associated with impaired crosslinking in granulation tissue; (3) chronic caloric restriction is associated with decreased collagen production and decreased levels of DHLNL; (4) the adverse effects of chronic caloric restriction on wound healing may have a greater impact on wound healing in old animals.

Interstitial pulmonary fibrosis induced by intratracheal instillation of bleomycin (BL) involves an excess production of reactive oxygen species, unavailability of adequate levels of NAD and ATP to repair the injured pulmonary epithelium, and an overexuberant lung collagen reactivity followed by deposition of highly cross-linked mature collagen fibrils resistant to enzymatic degradation. In the present study, we have demonstrated that dietary supplementation with taurine and niacin offered almost complete protection against the lung fibrosis in a multidose BL hamster model. The mechanisms for the protective effect of taurine and niacin are multifaceted. These include the ability of taurine to scavenge HOCl and stabilize the biomembrane; niacin's ability to replenish the BL-induced depletion of NAD and ATP; and the combined effect of taurine and niacin to suppress all aspects of BL-induced increases in the lung collagen reactivity, a hallmark of interstitial pulmonary fibrosis. It was concluded from the data presented at this Conference that the combined treatment with taurine and niacin, which offers a multipronged approach, will have great therapeutic potential in the intervention of the development of chemically induced interstitial lung fibrosis in animals and humans. Images Figure 4. A Figure 4. B

Understanding factors that promote pulmonary tolerance to long-term oxidant injury is essential to evaluating health risks in humans. One such factor may be Clara cell 10-kDa protein (CC10), a protein secreted by nonciliated cells in distal conducting airways thought to have an anti-inflammatory action against inhaled xenobiotics. Using standard immunohistochemical techniques and laser scanning confocal microscopy, we assessed changes in CC10 abundance in the centriacinar region of the rat following 20 months′ exposure to 0.12 and 1.00 ppm ozone. Three zones of reflectance intensity (high, medium, and low), directly related to CC10 density, were used to distinguish between the two major subcellular compartments where CC10 is distributed: granules and endoplasmic reticulum. Low levels of ozone (0.12 ppm) had no significant effect on the cellular distribution or abundance of CC10 in nonciliated epithelium in the centriacinar region. In contrast, 1.00 ppm ozone not only elevated cellular volume of granule-based CC10, but also elevated the protein′s concentration within the granules and increased the number of granules per cell profile. The proportion of nonciliated cells in terminal bronchioles increased significantly at the expense of ciliated cells. This combination of factors led to a threefold increase in CC10 stored per unit surface area of epithelium in terminal bronchioles. The nonciliated cells in ozone-induced respiratory bronchioles contained a distribution of CC10 similar to that of bronchiolar nonciliated cells in control animals. We conclude that ozone-induced tolerance may be related to the increased abundance and wider distribution of CC10 in central acini of rats following long-term ozone exposure.

Standard immunohistochemical techniques and laser scanning confocal microscopy were used to assess changes in the abundance of Clara cell 10 kD protein (CC10) within granules and endoplasmic reticulum of rat nonciliated cells after the administration of the secretagogue pilocarpine. Intracellular pools of CC10 were compared over time with time-matched controls at two airway levels, proximal bronchi and terminal bronchioles. Three zones of reflectance intensity (high, medium, and low), corresponding to different densities of CC10 were used to quantify changes in CC10 levels. We observed a shift in the abundance of CC10 from endoplasmic reticulum to granules 30 min after injection of pilocarpine. In addition, the depletion of granule-based CC10 occurred earlier in bronchial cells (30 min) than in bronchiolar cells (60 min). We also noted that the density of CC10 within the two cellular compartments remained steady even though the levels of CC10 dropped due to degranulation. Following degranulation, CC10 levels returned to near-control levels. In the absence of pilocarpine, the percentage of cell volume occupied by the granule-based CC10 indicated that two varieties of nonciliated cells exist in bronchial airways. However, administration of pilocarpine abolishes this difference in CC10 abundance. Our results demonstrate that bronchial cells respond sooner to the secretagogue than do their bronchiolar counterparts and/or possess biosynthetic capabilities that are more easily overwhelmed than those of the terminal bronchiolar cells. These differences suggest that proximal nonciliated cells are distinct from distal nonciliated cells.

Mitochondrial function has been linked to protection from and symptom reduction in chronic diseases such as heart disease, diabetes and metabolic syndrome. We review a number of phytochemicals and biofactors that influence mitochondrial function and oxidative metabolism. These include resveratrol found in grapes; several plant-derived flavonoids (quercetin, epicatechin, catechin and procyanidins); and two tyrosine-derived quinones, hydroxytyrosol in olive oil and pyrroloquinoline quinone, a minor but ubiquitous component of plant and animal tissues. In plants, these biofactors serve as pigments, phytoalexins or growth factors. In animals, positive nutritional and physiological attributes have been established for each, particularly with respect to their ability to affect energy metabolism, cell signaling and mitochondrial function.