Two Allelic Variants of Aldo-Keto Reductase 1A1 Exhibit Reduced in Vitro Metabolism of Daunorubicin

ABSTRACT: Aldo-keto reductases (AKRs) are a class of NADPH-dependent oxidoreductases that have been linked to metabolism of the anthracyclines doxorubicin (DOX) and daunorubicin (DAUN). Although widely used, cardiotoxicity continues to be a serious side effect that may be linked to metabolites or reactive intermediates generated in their metabolism. In this study we examine the little known effects of nonsynonymous single nucleotide polymorphisms of human AKR1A1 on the metabolism of these drugs to their alcohol metabolites. Expressed and purified from bacteria using affinity chromatography, the AKR1A1 protein with a single histidine (6x-His) tag exhibited the greatest activity using two test substrates: p-nitrobenzaldehyde (5.09 ؎ 0.16 mol/min/mg of purified protein) and DL-glyceraldehyde (1.24 ؎ 0.17 mol/min/mg). These activities are in agreement with published literature values of nontagged human AKR1A1. The 6x-His-tagged AKR1A1 wild type and allelic variants, E55D and N52S, were subsequently examined for metabolic activity using DAUN and DOX. The tagged variants showed significantly reduced activities (1.10 ؎ 0.42 and 0.72 ؎ 0.47 nmol of daunorubicinol (DAUNol) formed/min/mg of purified protein for E55D and N52S, respectively) compared with the wild type (2.34 ؎ 0.71 nmol/min/mg). The wild type and E55D variant metabolized DOX to doxorubicinol (DOXol); however, the levels fell below the limit of quantitation (25 nM). The N52S variant yielded no detectable DOXol. A kinetic analysis of the DAUN reductase activities revealed that both amino acid substitutions lead to reduced substrate affinity, measured as significant increases in the measured K m for the reduction reaction by AKR1A1. Hence, it is possible that these allelic variants can act as genetic biomarkers for the clinical development of DAUN-induced cardiotoxicity

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae

Mechanisms Underlying Stage-1 TRPL Channel Translocation in Drosophila Photoreceptors

Background: TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere), TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels. Methodology/Principal Findings: We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content. Conclusions/Significance: Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protei

Biomarkers for Severity of Spinal Cord Injury in the Cerebrospinal Fluid of Rats

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage

Etudes du developpement de la drosophile: variations on a theme

As a holometabolous organism which has been highly exploited genetically, the fruitfly, Drosophila, is an excellent model for the study of development in higher organisms. Conditional mutations that are sensitive to temperature differences were used to investigate problems of gene action in different tissues, regulation of specific tissue determination and differentiation, and the genetic regulation of development and functional integration of the nervous system. The first problem, a classical question in developmental biology, attempts to determine whether control of the activity of a structural gene is directly imparted by the tissue in which the gene product is active or whether, in fact, a freely flowing evocator of gene action exists. A temperature-sensitive ras allele has an effective lethal phase at 29°C about 12 hours after pupation and a temperature-sensitive period (TSP) for lethality beginning midway through the third larval instar and ending around pupation. The mutation also alters the quantity of pteridines present in the eyes, testes and malpighian tubules at 29°C. The TSP for pigment production in the malpighian tubules occurs in the egg and first instar larvae, and in eyes and testes after pupation. The demonstrated autonomy of the mutant in the eyes implies the tissue-specific functioning of the gene. It is suggested that the different TSPs for a single mutation indicate tissue-specific activation of a gene at different times during development, although the possibility of activation of preformed polypeptides has not been eliminated. There exists in Drosophila a class of mutations called "homeotic" which cause changes in determination of the imaginal discs. The second problem investigated concerns the possibility of isolating a temperature-sensive homeotic mutant for the purpose of studying genes which regulate specific pathways of differentiation. The homeotic mutant, ss[sup a40a], was found to have a temperature-sensitive transformation of the arista segment of the antennal complex to a tarsus of the leg. In a selected stock, penetrance was complete so that at 29°C, normal aristae were produced, whereas at 17°C, complete tarsi developed in all flies. "Shift" studies revealed a temperature-sensitive period in the third larval instar. The temperature-initiated action of ss[sup a40a] does not appear to act on a ts receptor site within the disc cells. In combination with another homeotic mutant, Antenna-pedia, the entire antennal complex is transformed to a complete leg at 22°C. Mutants affecting the nervous system were sought for the purpose of investigating the genetic regulation of the development and function of the nervous system. A temperature-sensitive mutation, para[sup ts] of Drosophila melanogaster causes an immediate but reversible paralysis only of adults when shifted from 22°C to 29°C. The mutation is a sex-linked recessive mapping 2.8 units to the left of f. Wild-type flies observed for two hour periods exhibited normal mobility at all temperatures between 22° and 35°C. From 22° to 25°C, para[sup ts] flies were wild-type in walking, climbing and flying ability. At one degree intervals above 25°C, para[sup ts] flies became increasingly debilitated and at 29°C, complete paralysis occurred. After prolonged maintenance at 29°C, recovery of some activity could occur at that temperature. Extensive studies of behavior of mosaics at 29°C revealed a requirement of the + allele in the head for mobility and a thoracic component for proper leg movement. Normal electroretino-grams were obtained at both 22° and 30°C. The results suggest a temperature-sensitive defect in the central nervous system.Science, Faculty ofZoology, Department ofGraduat