Erwin T. Cabacungan

The role of glutamine and other alternate substrates as energy sources in the fetal rat lung type II cell.

Glucose has been thought to be the primary substrate for energy metabolism in the developing lung; however, alternate substrates are used for energy metabolism in other organs. To examine the role of alternate substrates in the lung, we measured rates of oxidation of glutamine, glucose, lactate, and 3-hydroxybutyrate in type II pneumocytes isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates. Glutamine had a rate of 24.36 ± 4.51 nmol 14CO2 produced/h/mg of protein (mean ± SEM), whereas lactate had a significantly higher rate, 40.29 ± 4.42. 3-Hydroxybutyrate had a rate of 14.91 ± 1.93. The rate of glucose oxidation was 2.13 ± 0.36, significantly lower than that of glutamine. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate. This identifies multiple metabolic compartments of energy metabolism. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity, rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition. Our data suggest at least two separate compartments in the type II cells for substrate oxidation, one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung type II pneumocyte. In addition, we have delineated some of the compartmentation that occurs within the developing type II cell, which may determine how these substrates are used.

Differences in FMO2*1 allelic frequency between Hispanics of Puerto Rican and Mexican descent

A polymorphism for the phase I drug-metabolizing enzyme, flavin-containing monooxygenase isoform 2 (FMO2), encoding either truncated inactive protein, FMO2X472 (FMO2.2A), or full-length active enzyme, FMO2Q472 (FMO2.1), is known and exhibits significant interethnic differences in allelic frequency. FMO2 is the major or sole FMO isoform expressed in the lung of most mammals, including nonhuman primates. To date, FMO2.1 has been found only in African-American and Hispanic populations, rendering individuals with this allele subject to drug metabolism that is potentially different from that of the general population. Approximately 26% of African-Americans (n = 180) possess the FMO2*1 allele. In preliminary studies, we initially estimated that 5% of Hispanics (n = 40) have the FMO2*1 allele, but access to large cohorts of individuals of defined national origin has allowed us to determine the occurrence among Mexican-American and Puerto Rican-American groups. We used allele-specific genotyping to detect FMO2*1 from 632 Hispanic individuals, including 280 individuals of Mexican origin and 327 individuals of Puerto Rican origin. Statistical analysis indicated that results from Mexican (five sample sources) and Puerto Rican (three sample sources) samples were consistent with the hypothesis of homogeneity within each group from different sources. Data were subsequently pooled across sources to test for evidence of a difference in occurrence of FMO2*1 between ethnic groups. There was strong evidence (p = 0.0066) that FMO2*1 is more common among Puerto Ricans (7%) than among individuals of Mexican descent (2%). The overall occurrence of FMO2*1 among Hispanics of all origins is estimated to be between 2 and 7%.

Novel CYP2C9 promoter variants and assessment of their impact on gene expression

There are a considerable number of reports identifying and characterizing genetic variants within the CYP2C9 coding region. Much less is known about polymorphic promoter sequences that also might contribute to interindividual differences in CYP2C9 expression. To address this problem, approximately 10,000 base pairs of CYP2C9 upstream information were resequenced using 24 DNA samples from the Coriell Polymorphism Discovery Resource. Thirty-one single-nucleotide polymorphisms (SNPs) were identified; nine SNPs were novel, whereas 22 were reported previously. Using both sequencing and multiplex single-base extension, individual SNP frequencies were determined in 193 DNA samples obtained from unrelated, self-reported Hispanic Americans of Mexican descent, and they were compared with similar data obtained from a non-Latino white cohort. Significant interethnic differences were observed in several SNP frequencies, some of which seemed unique to the Hispanic population. Analysis using PHASE 2.1 inferred nine common (>1%) variant haplotypes, two of which included the g.3608C>T (R144C) CYP2C9*2 and two the g.42614A>C (I359L) CYP2C9*3 SNPs. Haplotype variants were introduced into a CYP2C9/luciferase reporter plasmid using site-directed mutagenesis, and the impact of the variants on promoter activity assessed by transient expression in HepG2 cells. Both constitutive and pregnane X receptor-mediated inducible activities were measured. Haplotypes 1B, 3A, and 3B each exhibited a 65% decrease in constitutive promoter activity relative to the reference haplotype. Haplotypes 1D and 3B exhibited a 50% decrease and a 40% increase in induced promoter activity, respectively. These data suggest that genetic variation within CYP2C9 regulatory sequences is likely to contribute to differences in CYP2C9 phenotype both within and among different populations.

Haplotype and functional analysis of four flavin-containing monooxygenase isoform 2 (FMO2) polymorphisms in Hispanics

Objectives Previous work defined two flavin-containing monooxygenase 2 (FMO2) alleles. The major allele, FMO2*2 (g.23,238C>T), encodes truncated inactive protein (p.X472) whereas the minor allele, FMO2*1, present in African- and Hispanic-American populations, encodes active protein (p.Q472). Recently, four common (27 to 51% incidence) FMO2 single nucleotide polymorphisms (SNPs) were detected in African-Americans (N=50); they encode the following protein variants: p.71Ddup, p.V113fs, p.S195L and p.N413 K. Our objectives were to: (1) determine the incidence of these SNPs in 29 Hispanic individuals previously genotyped as g.23,238C (p.Q472) and 124 previously genotyped as homozygous g.23,238 T (p.X472); (2) determine FMO2 haplotypes in this population; and (3) assess the functional impact of SNPs in expressed proteins. Methods SNPs were detected via allele-specific oligonucleotide amplification coupled with real-time or electrophoretic product detection, or single strand conformation polymorphism. Results The g.7,700_7,702dupGAC SNP (p.71Ddup) was absent. The remaining SNPs were present but, except for g.13,732C>T (p.S195L), were less common in the current Hispanic study population versus the previously described African-Americans. Only expressed p.N413 K was as active as p.Q472, as determined by methimazole- and ethylenethiourea-dependent oxidation. Haplotype determination demonstrated that the g.10,951delG (p.V113fs), g.13,732C>T (p.S195L) and g.22,060T>G (p.N413 K) variants segregated with g.23,238C>T (p.X472). Conclusions SNPs would not alter FMO2 activity in individuals possessing at least one FMO2*1 allele. It is likely that these SNPs will segregate similarly in African-American populations. Therefore, estimates that 26% of African-Americans and 2–7% of Hispanic-Americans have at least one FMO2*1 allele should closely reflect the percentages producing active FMO2 protein.

Genotyping and site-directed mutagenesis of a cytochrome P450 meander Pro-X-Arg motif critical to CYP4B1 catalysis

CYP4B1 isoforms from rodents and other common laboratory animals are involved in the bioactivation of a range of protoxins, including 2-aminofluorene, 4-ipomeanol, and valproic acid. However, an earlier study provided evidence for a human allele encoding a nonfunctional CYP4B1 enzyme due to a Pro427Ser transversion in the meander region of the protein. In the present study, the CYP4B1 gene from several racial groups, Caucasians, African-Americans, and Hispanics, and from six nonhuman primate species was genotyped using a PCR-Hinf1 restriction enzyme fragment length polymorphism assay or by direct sequencing. All human populations examined were found to possess only the Ser allele at codon 427 ((1279)TCT) and all of the nonhuman primate species possessed only the Pro (CCT) allele. Therefore, an inactivating (1279)C-->T mutation in the human CYP4B1 gene likely arose following divergence of the Homo and Pan clades. Amino acid sequence alignments revealed further that this key Pro residue is located two amino acid residues N-terminal to the distal Arg of a Glu-Arg-Arg triad thought to participate in heme binding and/or redox partner interactions. Mutation of the corresponding Arg424 residue in rabbit CYP4B1 to Leu, but not His, resulted in a loss of lauric acid hydroxylase activity and ability to generate a reduced-CO binding spectrum. These data provide additional evidence for the importance of this meander region Pro-X-Arg motif in CYP4B1 heme binding and catalytic function.

DEVELOPMENTALLY INDUCED CHANGES IN RAT LUNG MALIC ENZYME ACTIVITIES

To determine lung malic enzyme activity at varying stages of development, both cytosolic and mitochondrial enzyme activities were assayed in rat lungs at various stages from day 16 of fetal life to 2 months of postnatal life by measuring the production of 14 CO 2 from 14 C-malate. Malic enzyme activities were significantly higher in the mitochondrial than in the cytosolic fractions at all ages studied. The mitochondrial malic enzyme activity was significantly higher in canalicular stage (days 19-20) stage of lung development when compared to the glandular stage (days 16-18). The mitochondrial fraction at day 19 exhibited biphasic kinetics: high affinity, K m = 0.45 mmol, V max = 10.04 nmol/mg protein/min; and low affinity, K m = 5.48 mmol, V max = 56.83 nmol/mg protein/min. The cytosolic malic enzyme activity of all fetal stages (saccular stage [days 16-18], canalicular stage [days 19-20], and glandular stage [days 21-22] were significantly higher when compared to postnatal levels (postnatal days 1-10, adult). In contrast to the mitochondrial fraction, at day 19, the cytosolic fraction showed a single K m of 0.23 mmol, V max = 12.32 nmol/mg protein/min. The increased mitochondrialmalic enzyme activity during late gestation would suggest that, as we have previously demonstrated, anaplerotic substrates other than glucose, may provide a significant energy source in fetal lung. The increased cytosolic activity in the prenatal phases would suggest that the NADPH provided from malic enzyme is an important contributor to de novo fatty acid synthesis, leading to surfactant synthesis, critical to normal lung development in late gestation.To determine lung malic enzyme activity at varying stages of development, both cytosolic and mitochondrial enzyme activities were assayed in rat lungs at various stages from day 16 of fetal life to 2 months of postnatal life by measuring the production of 14CO2 from 14C-malate. Malic enzyme activities were significantly higher in the mitochondrial than in the cytosolic fractions at all ages studied. The mitochondrial malic enzyme activity was significantly higher in canalicular stage (days 19-20) stage of lung development when compared to the glandular stage (days 16-18). The mitochondrial fraction at day 19 exhibited biphasic kinetics: high affinity, Km = 0.45 mmol, Vmax = 10.04 nmol/mg protein/min; and low affinity, Km = 5.48 mmol, Vmax = 56.83 nmol/mg protein/min. The cytosolic malic enzyme activity of all fetal stages (saccular stage [days 16-18], canalicular stage [days 19-20], and glandular stage [days 21-22] were significantly higher when compared to postnatal levels (postnatal days 1-10, adult). In contrast to the mitochondrial fraction, at day 19, the cytosolic fraction showed a single Km of 0.23 mmol, Vmax = 12.32 nmol/mg protein/min. The increased mitochondrial malic enzyme activity during late gestation would suggest that, as we have previously demonstrated, anaplerotic substrates other than glucose, may provide a significant energy source in fetal lung. The increased cytosolic activity in the prenatal phases would suggest that the NADPH provided from malic enzyme is an important contributor to de novo fatty acid synthesis, leading to surfactant synthesis, critical to normal lung development in late gestation.

Pseudohypoaldosteronism Type 1 (arPHA1) Treated With Sodium Polystyrene Sulfonate Pretreated Milk

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