Pyrroloquinoline quinone nutritional status alters lysine metabolism and modulates mitochondrial DNA content in the mouse and rat.

Pyrroloquinoline quinone (PQQ) added to purified diets devoid of PQQ improves indices of perinatal development in rats and mice. Herein, PQQ nutritional status and lysine metabolism are described, prompted by a report that PQQ functions as a vitamin-like enzymatic cofactor important in lysine metabolism (Nature 422 [2003] 832). Alternatively, we propose that PQQ influences lysine metabolism, but by mechanisms that more likely involve changes in mitochondrial content. PQQ deprivation in both rats and mice resulted in a decrease in mitochondrial content. In rats, alpha-aminoadipic acid (alphaAA), which is derived from alpha-aminoadipic semialdehyde (alphaAAS) and made from lysine in mitochondria, and the plasma levels of amino acids known to be oxidized in mitochondria (e.g., Thr, Ser, and Gly) were correlated with changes in the liver mitochondrial content of PQQ-deprived rats, but not PQQ-supplemented rats. In contrast, the levels of NAD dependent alpha-aminoadipate-delta-semialdehyde dehydrogenase (AASDH), a cytosolic enzyme important to alphaAA production from alphaAAS, was not influenced by PQQ dietary status. Moreover, the levels of U26 mRNA were not significantly changed even when diets differed markedly in PQQ and dietary lysine content. U26 mRNA levels were measured, because of U26's proposed, albeit questionable role as a PQQ-dependent enzyme involved in alphaAA formation.

Characterization of a novel airway epithelial cell-specific short chain alcohol dehydrogenase/reductase gene whose expression is up-regulated by retinoids and is involved in the metabolism of retinol.

Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situ hybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus.

Retinol binding protein expression is induced in HepG2 cells by zinc deficiency.

Zinc (Zn) deficiency is often associated with low plasma vitamin A (retinol) concentrations. It has been suggested that the reduction in plasma retinol is secondary to reduced liver retinol binding protein (RBP) synthesis. In the present study, RBP expression was determined in HepG2 cells cultured in either Zn adequate media or chelated media containing varying concentrations of Zn. Levels of RBP mRNA increased in a time- and Zn concentration-dependent manner such that 0.5 microM Zn-treated cells exhibited a >7.5-fold increase while cells treated with 15 microM Zn were increased 2.9-fold at 72 h compared to controls. RBP protein also progressively increased by 72 h to levels >8-fold and 3-fold higher than controls, in 0.5 microM and 15 microM Zn-treated cells, respectively. The increase in RBP occurred without any change in DNA concentration between groups through 72 h. The Zn deficiency-induced elevations in RBP transcript levels could be reversed within 24-48 h of repletion in Zn adequate media. Thus, the reductions in plasma retinol observed in Zn deficiency are in part a direct consequence of the deficiency.

Metabolic conversion of retinol to retinoic acid mediates the biological responsiveness of human mammary epithelial cells to retinol.

The biological effects of vitamin A are mediated in part by retinoic acid (RA) modulation of gene transcription. In this study, we examined whether normal human mammary epithelial cells (HMECs) are biologically responsive to retinol (ROH), the metabolic precursor of RA. While both ROH and tRA resulted in time- and dose-dependent decreases in total cell number, tRA was markedly more potent. Metabolically, treatment of HMECs with physiological doses of ROH resulted in rapid uptake and subsequent production of both retinyl esters and tRA. Although a comparatively minor metabolite, tRA levels peaked at 6 h and remained above endogenous levels for up to 72 h in proportion to cellular ROH concentrations. In HMECs transfected with an RA-responsive luciferase reporter gene, treatment with 3 microM ROH resulted in an increase in luciferase activity to a level intermediate between that observed with 0.001 and 0.01 microM tRA. Citral, an RA-synthesis inhibitor, was also used to examine the biological activity of ROH. Compared to ROH alone, ROH plus citral treatment resulted in three-fold less tRA synthesis and a > 65% attentuation of RA-responsive reporter gene activity which persisted through 72 h. Citral also significantly attenuated the extent of ROH-mediated reductions in total HMEC number. Thus, treatment with physiological concentrations of ROH results in fewer total numbers of HMECs and this response is a consequence of cellular tRA synthesis which can induce RA-responsive gene expression.

Mouse retinol binding protein gene: cloning, expression and regulation by retinoic acid.

A full-length cDNA clone encoding the retinol binding protein (RBP) was isolated from a mouse liver cDNA library by hybridization screening. The nucleotide sequence of murine RBP is 85 and 95% homologous to that of human and rat RBP, respectively, with a deduced amino acid sequence > or = 83% homologous to both species. Analysis of the tissue expression pattern of RBP mRNA in the female mouse indicated relatively abundant expression in the liver, with lesser amounts in extrahepatic tissues including adipose, kidney, spleen and uterus, suggesting that these tissues may have a significant role in retinol homeostasis. Mouse liver cell RBP regulation by retinoids was also investigated. Both all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9c-RA) induced RBP mRNA expression in a dose- and time-dependent manner. Maximal levels (up to 4-fold above controls) were observed at > or = 48 h following treatment of both mouse hepatoma cells in vitro and in vivo in mice receiving a single, oral dose of either retinoid. Interestingly, 9c-RA was more potent at RBP induction in both in vivo and in vitro systems. Given the extent and temporal pattern of RBP induction, we suggest that the RA-mediated increase in liver RBP is part of a cellular protection mechanism. Increased levels of RBP would facilitate sequestration and possibly cellular export of RA in cells receiving prolonged exposure to high levels of RA, thus minimizing toxicity.

Induction of mouse retinol binding protein gene expression by cyclic AMP in Hepa 1-6 cells.

Retinol binding protein (RBP) is the primary circulating transport molecule for retinol, facilitating its transport to target tissues and influencing target cell uptake. Specific signals and molecular mechanisms that regulate RBP gene expression are poorly understood. Using the mouse hepatoma cell line (Hepa 1-6), we examined the role of cAMP in the molecular regulation of RBP. Dibutyryl cAMP (dbcAMP) or the adenylate cyclase activator, forskolin, increased RBP mRNA levels >6-fold at 24 h. Increases in RBP mRNA were dose dependent over the range of 10 microM-1 mM for dbcAMP and 0.5-10 microM for forskolin. 8-Bromo cAMP, a nonhydrolyzable analog, over the range of 0.01-0.5 mM, increased RBP mRNA levels 9.2-fold at 24 h. Induction of RBP transcripts by analogs also resulted in a comparable increase in intracellular RBP protein. Cycloheximide (10 microgram/ml) did not prevent cAMP-mediated induction of RBP mRNA, indicating that de novo protein synthesis is not required for cAMP-mediated induction of RBP transcription. These studies demonstrate that cAMP, or agents which elevate intracellular cAMP, increase RBP transcript levels. The time course and extent of RBP mRNA induction and the resultant increase in RBP protein support the concept that cAMP regulation of RBP gene expression may be physiologically relevent. Given the ubiquitous nature of cAMP as a second messenger, and the several mechanisms by which cAMP regulates gene expression, studies are in progress to define molecular mechanisms by which cAMP regulates RBP gene expression.

Cloning and biological characterization of human single-chain Fv fragments that mediate neutralization of HIV-1.

OBJECTIVE: To develop recombinant single-chain Fv fragments against HIV-1 gp120. METHODS: A panel of human monoclonal antibody Fv fragments were generated against the HIV-1 gp120 by affinity selection from an antibody library expressed on the surface of filamentous phage. The library was prepared from peripheral blood lymphocytes of an asymptomatic HIV-1-infected mother with a high neutralization titer. This mother did not transmit HIV-1 to her offspring (non-transmitter). Heavy and light chains were initially amplified separately and combined by splicing by overlap extension to generate Fv fragments. RESULTS: Several clones expressing single-chain Fv fragments bind strongly to HIV-1 gp120 and several were found to neutralize cell-free HIV-1IIIB. Gross epitope mapping suggest that different clones bound to different functional regions on the envelope. The clones also exhibited sequence diversity. CONCLUSIONS: This strategy of cloning resulted in the development of functional human-derived antibody reagents with different anti-HIV-1 biological properties in vitro. These recombinant Fv fragments have potential utility as immune reagents, as well as in the design of potential immunotherapeutics. In addition, these antibody reagents may provide information on the relationship between humoral immunity and maternal-fetal (vertical) HIV-1 transmission.

The complete structure of human class IV alcohol dehydrogenase (retinol dehydrogenase) determined from the ADH7 gene.

A novel human alcohol dehydrogenase (ADH) gene called ADH7 has been characterized and determined to encode class IV ADH, an ADH isozyme which is very active as a retinol dehydrogenase. A nearly full-length cDNA for ADH7 was isolated from a human stomach cDNA library, and a 5' genomic clone containing exons 1 and 2 was isolated from a human genomic library. DNA sequence analysis of the cDNA and genomic clones revealed the complete coding region of the gene and the deduced full-length amino acid sequence of human class IV ADH composed of 373 amino acids following the initiator methionine. The class IV identity of the sequence was confirmed by agreement with previously determined sequences for several human stomach class IV ADH peptides. Alignment of the full-length predicted amino acid sequence of human class IV ADH with the full-length sequences of the other four known human ADH classes revealed sequence identities of 69% (class I), 59% (class II), 61% (class III), and 60% (class V). The higher sequence identity shared with human class I ADH suggests that the genes for ADH classes I and IV may have diverged from a common ancestor after the separation of the other classes, and may still share common physiological functions. Discussed is the possibility that one of these functions is retinol oxidation for the synthesis of retinoic acid, a hormone important for cellular differentiation.

Differential activity of the promoter for the human alcohol dehydrogenase (retinol dehydrogenase) gene ADH3 in neural tube of transgenic mouse embryos.

Mammalian alcohol dehydrogenase (ADH) has previously been shown to function in vitro as a retinol dehydrogenase as well as an ethanol dehydrogenase. Thus ADH participates in the conversion of retinol (vitamin A alcohol) to retinoic acid, a regulatory ligand for the retinoic acid receptor class of transcription factors. Human ADH exists as a family of isozymes encoded by seven genes, which are differentially expressed in adult liver and extrahepatic tissues, being found preferentially in the epithelial cells which are retinoid target tissues. However, human ADH expression patterns have not been analyzed in early embryonic tissues, which are known to synthesize and respond to retinoic acid such as the neural tube and limb buds. To estimate the embryonic expression pattern for one member of the human ADH family, we have constructed transgenic mouse lines carrying the human ADH3 promoter fused to the lacZ gene. ADH3-lacZ transgene expression was first noted at embryonic day 9.5 and was active in the neural tube extending from the midbrain to the spinal cord, as well as the heart and proximal regions of the forelimb buds. In day 12.5 and 13.5 embryos, ADH3 transgene expression remained in the neural tube and heart and was also observed in more distal regions of the forelimb and hindlimb buds as well as the kidney. Expression in the neural tube was highest in the ventral midline including the floor plate and showed a ventral to dorsal gradient of decreasing expression. These findings indicate that at least one human ADH isozyme may exist in the correct tissues to act as an embryonic retinol dehydrogenase catalyzing the synthesis of retinoic acid.

Developmental changes in endogenous retinoids during pregnancy and embryogenesis in the mouse.

Vitamin A and its analogs (retinoids) have acquired particular significance in embryonic development since the discovery that retinoic acid (RA) possesses properties of an endogenous morphogen and that embryonic tissues contain specific nuclear receptors for RA. Since the mammalian embryo does not synthesize RA de novo but rather must acquire it directly or in a precursor form from the maternal circulation, we sought to establish the relationship between levels of RA, retinol, and retinyl esters in the maternal system and their acquisition by the embryo, particularly during organogenesis in the mouse. Results indicate profound changes in maternal vitamin A levels during pregnancy in the mouse. These changes were characterized by a large, transient decrease in plasma retinol levels coincident with the period of organogenesis (e.g. gestational Days 9-14), and an apparent increase in mobilization from hepatic stores to the conceptus. During organogenesis, the embryo exhibited a steady increase in retinol levels with little increase in retinyl esters and virtually no change in RA. Analysis of retinoid accumulation patterns in the embryonic liver indicate that functional onset of vitamin A storage occurs by mid-organogenesis. In contrast, placental levels of these retinoids remained unchanged throughout organogenesis. Analysis of the conceptus as a developmental unit revealed that during early organogenesis the majority of retinoids are contained in the placenta (8-fold more than in the embryo). However, by mid-organogenesis the retinoid content of the embryo exceeds that of the placenta. Together, these results provide evidence that pregnancy in the mouse is accompanied by pronounced alterations in maternal retinoid homeostasis that occur coincident with the period of high embryonic sensitivity to exogenous retinoids.

Elevations in the endogenous levels of the putative morphogen retinoic acid in embryonic mouse limb-buds associated with limb dysmorphogenesis.

Retinoic acid, an endogenous metabolite of vitamin A (retinol), possesses striking biological activity akin to a morphogen in developing and regenerating vertebrate limbs. Systemic administration of retinoic acid (RA) to pregnant mammals during the period of limb organogenesis invariably results in dose-dependent dysmorphogenesis. In an attempt to uncover the mode of action of RA in the developing limb bud we analyzed, by HPLC methods, the levels of RA and its metabolic precursor, retinol, in embryonic mouse tissues prior to and following maternal exposure to a teratogenic dose of RA. Detectable levels of both RA and its isomer 13-cis-retinoic acid were found in the limb buds of Day 11 mouse embryos (40 +/- 2 somites). Although retinol was the major retinoid found in ethanolic extracts of either whole embryo or the limb buds, the latter is enriched in RA compared to the whole embryo. This indicated either a higher degree of retinol metabolism or a sequestration of RA in the limb bud compared to the rest of the embryo at this stage of development. A study of the time course of retinoid levels in treated embryos showed that changes occur rapidly, are stable for several hours, and then begin to return to pretreatment levels. After a maternal dose of 10 mg/kg RA, which resulted in a mild degree of limb anomalies, peak RA levels in the limb bud increased 50-fold over the endogenous level; a full 300-fold increase was found after a 100 mg/kg dose which results in 100% incidence of phocomelia. Interestingly, a dose-dependent depression in retinol levels was observed after RA treatment both in maternal plasma as well as the embryo. Studies are in progress to trace the intracellular disposition of both retinol and RA as well as any further active metabolite of RA in the limb buds and other embryonic tissues.

Pharmacokinetic assessment of teratologically effective concentrations of an endogenous retinoic acid metabolite.

Retinoic acid is a natural vitamin A derivative that undergoes oxidative metabolism in the body to yield several metabolites, which apparently represent the products of a detoxification pathway. To assess if such metabolic conversions diminished teratogenic potency, one of the major metabolites (4-oxo-all-trans-retinoic acid) was tested for its teratogenic activity in pregnant ICR mice and further investigated for its pharmacokinetic features to determine if it accumulated in the embryo in concentrations sufficient to elicit a teratogenic response. Administration of single oral doses (10, 25, 50, or 100 mg/kg) of the compound to ICR mice on day 11 of gestation (plug day = day 0) produced dose-dependent frequencies of serious fetal anomalies of the type usually associated with the use of retinoic acid and other retinoids. The metabolite was equivalent in teratogenic potency to retinoic acid, and, in the instance of cleft palate frequency, it was even more active. Concentrations of 4-oxo-all-trans-retinoic acid and its 13-cis isomer were measured in the maternal plasma and whole embryos at 30 min to 10 hr after administration of the lowest (10 mg/kg) and the highest (100 mg/kg) teratogenic dose of 4-oxo-all-trans-retinoic acid by means of high-performance liquid chromatography methodology. Distribution of the compound in the maternal system and transfer to the embryo occurred rapidly with either dose. Peak concentration in the maternal plasma and the embryo persisted for 3-4 hr after the higher dose but not with the lower dose; however, elimination kinetics for the two dose levels were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Derivation of retinoic acid and metabolites from a teratogenic dose of retinol (vitamin A) in mice.

Megadose supplements of vitamin A are under suspicion as hazards to the developing embryo after the discovery that two vitamin A-related drugs, Accutane and Tigason, are human teratogens. Retinoic acid (all-trans-RA) is a natural metabolite of vitamin A which participates in many of the known functions of vitamin A and may be the active agent in teratogenesis. In this investigation we gave a single, high oral dose of retinol (vitamin A) to pregnant mice to assess its transplacental pharmacokinetics as well as to measure the formation and distribution of its metabolites in the embryo. Retinol was estimated to be 4-fold less active than retinoic acid in the whole animal teratogenesis and 20-fold less active in the in vitro bioassay. A fully teratogenic dose, 200 mg/kg, yielded considerable quantities of retinoic acid which were transferred to the embryo with kinetics similar to that of retinol. During the first 8 hr after administration of retinol, the metabolites (including all-trans-RA, 13-cis-RA, and 4-oxo-RA) constituted almost 50% of the quantity of all retinol derivatives found in the embryo. A comparison of combined peak concentrations of the metabolites (or their AUC values) with the extent of teratogenesis associated with them individually provided sufficient evidence to implicate the metabolites themselves as mediators of retinol-induced teratogenesis. However, since both retinol and retinoic acid were present in sufficient concentrations in the embryo to act as teratogens we cannot at present rule out the possibility that they may act independently. Further experimentation will be necessary to address whether retinoic acid detected in the embryo is the product of the embryo's own metabolic capability or is transferred from the maternal circulation.