Clinical importance of risk variants in the dihydropyrimidine dehydrogenase gene for the prediction of early-onset fluoropyrimidine toxicity.

We investigated the clinical relevance of dihydropyrimidine dehydrogenase gene (DPYD) variants to predict severe early-onset fluoropyrimidine (FP) toxicity, in particular of a recently discovered haplotype hapB3 and a linked deep intronic splice site mutation c.1129-5923C>G. Selected regions of DPYD were sequenced in prospectively collected germline DNA of 500 patients receiving FP-based chemotherapy. Associations of DPYD variants and haplotypes with hematologic, gastrointestinal, infectious, and dermatologic toxicity in therapy cycles 1-2 and resulting FP-dose interventions (dose reduction, therapy delay or cessation) were analyzed accounting for clinical and demographic covariates. Fifteen additional cases with toxicity-related therapy delay or cessation were retrospectively examined for risk variants. The association of c.1129-5923C>G/hapB3 (4.6% carrier frequency) with severe toxicity was replicated in an independent prospective cohort. Overall, c.1129-5923G/hapB3 carriers showed a relative risk of 3.74 (RR, 95% CI = 2.30-6.09, p = 2 × 10(-5)) for severe toxicity (grades 3-5). Of 31 risk variant carriers (c.1129-5923C>G/hapB3, c.1679T>G, c.1905+1G>A or c.2846A>T), 11 (all with c.1129-5923C>G/hapB3) experienced severe toxicity (15% of 72 cases, RR = 2.73, 95% CI = 1.61-4.63, p = 5 × 10(-6)), and 16 carriers (55%) required FP-dose interventions. Seven of the 15 (47%) retrospective cases carried a risk variant. The c.1129-5923C>G/hapB3 variant is a major contributor to severe early-onset FP toxicity in Caucasian patients. This variant may substantially improve the identification of patients at risk of FP toxicity compared to established DPYD risk variants (c.1905+1G>A, c.1679T>G and c.2846A>T). Pre-therapeutic DPYD testing may prevent 20-30% of life-threatening or lethal episodes of FP toxicity in Caucasian patients.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.

The impact of testosterone, tibolone and black cohosh on purified mammary and placental 17β-hydroxysteroid dehydrogenase type 1

Abstract Context: Mammary and placental 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1). Objective: To assess the impact of testosterone, tibolone, and black cohosh on purified mammary and placental 17βHSD1. Materials and methods: 17βHSD1 was purified from human mammary gland and placenta by column chromatography, its activity was monitored by a radioactive activity assay, and the degree of purification was determined by gel electrophoresis. Photometric cofactor transformation analysis was performed to assess 17βHSD1 activity without or in presence of testosterone, tibolone and black cohosh. Results: 17βHSD1 from both sources displayed a comparable basal activity. Testosterone and tibolone metabolites inhibited purified mammary and placental 17βHSD1 activity to a different extent, whereas black cohosh had no impact. Discussion: Studies on purified enzymes reveal the individual action of drugs on local regulatory mechanisms thus helping to develop more targeted therapeutic intervention. Conclusion: Testosterone, tibolone and black cohosh display a beneficial effect on local mammary estrogen metabolism by not affecting or decreasing local estradiol exposure.

Lactate dehydrogenase concentration in nasal wash fluid indicates severity of rhinovirus-induced wheezy bronchitis in preschool children.

The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis.

Human 3β-hydroxysteroid dehydrogenase deficiency seems to affect fertility but may not harbor a tumor risk: lesson from an experiment of nature.

CONTEXT 3β-hydroxysteroid dehydrogenase deficiency (3βHSD) is a rare disorder of sexual development and steroidogenesis. There are two isozymes of 3βHSD, HSD3B1 and HSD3B2. Human mutations are known for the HSD3B2 gene which is expressed in the gonads and the adrenals. Little is known about testis histology, fertility and malignancy risk. OBJECTIVE To describe the molecular genetics, the steroid biochemistry, the (immuno-)histochemistry and the clinical implications of a loss-of-function HSD3B2 mutation. METHODS Biochemical, genetic and immunohistochemical investigations on human biomaterials. RESULTS A 46,XY boy presented at birth with severe undervirilization of the external genitalia. Steroid profiling showed low steroid production for mineralocorticoids, glucocorticoids and sex steroids with typical precursor metabolites for HSD3B2 deficiency. The genetic analysis of the HSD3B2 gene revealed a homozygous c.687del27 deletion. At pubertal age, he showed some virilization of the external genitalia and some sex steroid metabolites appeared likely through conversion of precursors secreted by the testis and converted by unaffected HSD3B1 in peripheral tissues. However, he also developed enlarged breasts through production of estrogens in the periphery. Testis histology in late puberty revealed primarily a Sertoli-cell-only pattern and only few tubules with arrested spermatogenesis, presence of few Leydig cells in stroma, but no neoplastic changes. CONCLUSIONS The testis with HSD3B2 deficiency due to the c.687del27 deletion does not express the defective protein. This patient is unlikely to be fertile and his risk for gonadal malignancy is low. Further studies are needed to obtain firm knowledge on malignancy risk for gonads harboring defects of androgen biosynthesis.

BIOCHEMICAL CHARACTERIZATION AND GENETIC MAPPING OF WILD TYPE AND MUTANT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ALLELES IN CHINESE HAMSTER OVARY CELLS

A UV-induced mutation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) was characterized in the CHO clone A24. The asymmetric 4-banded zymogram and an in vitro GAPD activity equal to that of wild type cells were not consistent with models of a mutant heterozygote producing equal amounts of wild type and either catalytically active or inactive mutant subunits that interacted randomly. Cumulative evidence indicated that the site of the mutation was the GAPD structural locus expressed in CHO wild type cells, and that the mutant allele coded for a subunit that differed from the wild type subunit in stability and kinetics. The evidence included the appearance of a fifth band, the putative mutant homotetramer, after addition of the substrate glyceraldehyde-3-phosphate (GAP) to the gel matrix; dilution experiments indicating stability differences between the subunits; experiments with subsaturating levels of GAP indicating differences in affinity for the substrate; GAPD zymograms of A24 x mouse hybrids that were consistent with the presence of two distinct A24 subunits; independent segregation of A24 wild type and mutant electrophoretic bands from the hybrids, which was inconsistent with models of mutation of a locus involved in posttranslational modification; the mapping of both wild type and mutant forms of GAPD to chromosome 8; and the failure to detect any evidence of posttranslational modification (of other A24 isozymes, or through mixing of homogenates of A24 and mouse).^ The extent of skewing of the zymogram toward the wild type band, and the unreduced in vitro activity were inconsistent with models based solely on differences in activity of the two subunits. Comparison of wild type homotetramer bands in wild type cells and A24 suggested the latter had a preponderance of wild type subunits over mutant subunits, and had more GAPD tetramers than did CHO controls.^ Two CHO linkages, GAPD-triose phosphate isomerase, and acid phosphatase 2-adenosine deaminase were reported provisionally, and several others were confirmed. ^

The identification of a 9-cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-cis retinoic acid

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.

Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5′ region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

In Azotobacter vinelandii, the E1 subunit of the pyruvate dehydrogenase complex binds fpr promoter region DNA and ferredoxin I

In Azotobacter vinelandii, deletion of the fdxA gene that encodes a well characterized seven-iron ferredoxin (FdI) is known to lead to overexpression of the FdI redox partner, NADPH:ferredoxin reductase (FPR). Previous studies have established that this is an oxidative stress response in which the fpr gene is transcriptionally activated to the same extent in response to either addition of the superoxide propagator paraquat to the cells or to fdxA deletion. In both cases, the activation occurs through a specific DNA sequence located upstream of the fpr gene. Here, we report the identification of the A. vinelandii protein that binds specifically to the paraquat activatable fpr promoter region as the E1 subunit of the pyruvate dehydrogenase complex (PDHE1), a central enzyme in aerobic respiration. Sequence analysis shows that PDHE1, which was not previously suspected to be a DNA-binding protein, has a helix–turn–helix motif. The data presented here further show that FdI binds specifically to the DNA-bound PDHE1.