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.

Chromosomal mapping and expression levels of a mouse soluble epoxide hydrolase gene.

The chromosomal location of a murine soluble epoxide hydrolase gene was determined using in situ mapping, restriction fragment length polymorphism (RFLP) and simple sequence length polymorphism (SSLP) analysis. In situ hybridization to mouse metaphase chromosomes using a soluble epoxide hydrolase cDNA probe showed that soluble epoxide hydrolase maps at band D of chromosome 14. An RFLP found between Mus castaneus (CAST) and Mus musculus (MEV) was used to map the soluble epoxide hydrolase gene in CAST x MEV intersubspecific testcross progeny to 14 cM from the Np-1 locus on mouse chromosome 14. SSLP markers were then used to confirm the location of soluble epoxide hydrolase at 14.0 +/- 3.7 cM distal to Np-1 and 19.2 +/- 4.3 cM proximal to D14Mit7. This region of mouse chromosome 14 is homologous with human chromosomes 8, 13 and 14. Enzyme assays and immunoblotting results suggest significant quantitative differences in expression of soluble epoxide hydrolase among three mouse strains. Northern blotting analysis showed that soluble epoxide hydrolase mRNA levels were correlated with the relative level of soluble epoxide hydrolase enzyme activity and soluble epoxide hydrolase protein in all three mouse strains.

Calcium buffering is required to maintain bone stiffness in saline solution.

This work determined whether mineral dissolution due to prolonged testing or storage of bone specimens in normal saline would alter their elastic modulus. In one experiment, small pieces of equine third metacarpal bone were soaked in normal saline supplemented with varying amounts of CaCl2. Changing Ca ion concentrations in the bath were monitored and the equilibrium concentration was determined. In a second experiment, the elastic moduli of twenty 4 x 10 x 100 mm equine third metacarpal beams were determined non-destructively in four-point bending. Half the beams were then soaked for 10 days in normal saline, and the other half in saline buffered to the bone mineral equilibrium point with Ca ions. Modulus measurements were repeated at 6 and 10 days. The equilibrium Ca ion concentration for bone specimens was found to be 57.5 mg l-1. The modulus of bone specimens soaked in normal saline significantly diminished 2.4%, whereas the modulus of those soaked in calcium-buffered saline did not change significantly.

Molecular cloning and expression of murine liver soluble epoxide hydrolase.

A clofibrate-induced mouse liver cDNA library was prepared and used to isolate the coding sequence for soluble epoxide hydrolase. A 1668-base pair (bp) clone was isolated and found to contain a 1269-bp open reading frame coding for 423 amino acids. Subsequent RNA polymerase chain reaction resulted in the isolation of 396 bp of additional 5'-sequence. Translation of the resulting 1659-bp open reading frame produced a 553-residue protein (62,527 Da) containing deduced peptide segments that matched the amino acid sequences of six peptide fragments isolated previously from CNBr digests of pure murine soluble epoxide hydrolase. Neither the DNA nor the protein sequence showed significant similarity to other currently published sequences. Structural analysis of the soluble epoxide hydrolase coding region suggested at least one potential regulatory motif. Expression of the composite cDNA in COS-7 cells resulted in a 5-10-fold increase in soluble epoxide hydrolase activity and a similar increase in soluble epoxide hydrolase protein amount compared to mock-transfected or vector control-transfected cells. Treatment of C57BL/6J mice with clofibrate led to an approximately 4-fold increase in both soluble epoxide hydrolase enzyme activity and steady-state mRNA levels.

Leukotoxin-diol: a putative toxic mediator involved in acute respiratory distress syndrome.

Leukotoxin is clinically associated with acute respiratory distress syndrome (ARDS). Recently, we found that leukotoxin-diol, the hydrated product of leukotoxin, is more toxic than the parent leukotoxin in vitro (Moghaddam and colleagues, Nature Med. 1997;3:562-566). To test if this difference in the toxicity of leukotoxin and leukotoxin-diol exists in vivo, Swiss Webster mice were administered leukotoxin or leukotoxin-diol. All mice treated with leukotoxin-diol died of ARDS-like respiratory distress, whereas the animals exposed to leukotoxin at the same dose survived. Histopathologic evaluation of the lungs revealed massive alveolar edema and hemorrhage with interstitial edema around blood vessels in the lungs of mice treated with leukotoxin-diol, whereas the lungs of mice treated with identical doses of leukotoxin had perivascular edema only and little change in alveolar spaces. Immunohistochemistry showed that the soluble epoxide hydrolase responsible for the hydrolysis of leukotoxin to its diol is concentrated in the vascular smooth muscle of small and medium-sized pulmonary vessels. In addition, 4-phenylchalcone oxide, an inhibitor of soluble epoxide hydrolase, was found to decrease the mortality induced by leukotoxin but had no effect on mortality induced by leukotoxin-diol. These studies provide strong in vivo evidence that leukotoxin may act as a protoxicant and that the corresponding diol is a putative toxic mediator involved in the development of ARDS.

Development of phase II xenobiotic metabolizing enzymes in differentiating murine clara cells.

Glutathione S-transferases (GSTs) and epoxide hydrolases (EHs) protect cells from exogenous insult by detoxifying electrophilic compounds. Little is known about these enzyme systems during postnatal lung development. This study was designed to help establish whether the heightened neonatal susceptibility of the lung to bioactivated cytotoxicants is the result of inadequate ability to detoxify reactive intermediates. We compared the distribution of immunoreactive protein and enzymatic activity of GSTs and EHs in isolated distal airways during pre- and postnatal development in lungs of mice from 16 days gestation to 9 weeks postnatal age (adult). GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels at postnatal day 28, and slightly lower levels in adults. Immunoreactive protein of isozyme pi has a peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity in distal airways increased with age. Microsomal EH protein expression increased in intensity with age, while activity was similar in airways from all ages. We conclude that in the mouse lung (1) cellular expression of glutathione S-transferase varies by age and isozyme and does not increase with increasing age, (2) airway glutathione S-transferase activity increases with increasing age and does not correlate with immunoreactive protein expression, and (3) airway microsomal epoxide hydrolase activity does not increase, even though immunoreactive protein expression does increase with age.